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Key value for chemical safety assessment

Effects on fertility

Description of key information

Under the conditions of the EOGRTS, Diammonium Peroxodisulphate (APS) caused death of pregnant female animals, clinical signs, reduced body weight and food consumption (male and female), changes in the gastro-intestinal tract – supported by histological findings – in dead pregnant female animals at 200/180 mg/kg bw/day of P generation. The copulatory and fertility ability of male and female animal were not selectively influenced at 200/180 mg/kg bw/day. There was a clearly lower number of dams delivered due to premature death related to severe maternal toxicity  at 200/180 mg/kg bw/day.
At 100 mg/kg bw/day of P generation, mortality of one dam, clinical signs, reduced body weight and food consumption (male and female), macroscopic and histopathological findings (only in dead animal) were observed. The copulatory and fertility ability (male or female animals) was considered normal. One pregnant female did not deliver due to premature death at 100 mg/kg bw/day.
At 50 mg/kg bw/day of P generation, there were no signs of systemic toxicity or influence on the reproductive performance (male or female animals)
In F1 offspring, reduced development (surface righting reflex, pinna detachment, eye opening) and depressed body weight development were observed at 100 and 200/180 mg/kg bw/day.
At 50 mg/kg bw/day of F1 offspring, no test item related effects were detected.
F1 adult (F1 Cohort 1A, Cohort 1B)
Reduced body weight development and food consumption were detected at 100 and 180 mg/kg bw/day in F1 adult animals (male and female). The degree of alterations at 100 mg/kg bw/day were minor and were considered to be toxicologically not relevant.
At 50 mg/kg bw/day of F1 adults, no test item related effects were detected.
Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for systemic toxicity (male and female): 50 mg/kg bw/day
NOAEL for reproductive performance (male): 180 mg/kg bw/day
NOAEL for reproductive performance (female): 50 mg/kg bw/day
NOAEL for F1 Offspring: 50 mg/kg bw/day
NOAEL for F1 Adults: 100 mg/kg bw/day
As all substances of the Persulfate Category share the same anionic persulfate moiety and similar toxicological properties a read across approach was applied for dipotassium persulfate and disodium persulfate using results obtained with diammonium persulfate in accordance the ECHA compliance check decisions (CCH-D-2114340955-43-01/F, CCH-D-2114340956-41-01/F, CCH D-2114340958-37-01/F).

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
DRF for OECD 443
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2019-11-20 to 2020-03-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29th July 2016
Deviations:
yes
Remarks:
DRF
GLP compliance:
no
Remarks:
This study was not performed according to GLP compliances, however, the principles of GLP were followed and all data were recorded and retained.
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as suitable species for toxicity and reproduction toxicity studies and the test guidelines were designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in toxicity and reproduction toxicity studies and wellknown fertility parameters.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt., Cserkesz u. 90., 1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 13-14 weeks
- Weight at study initiation: Males: 354-422 g; Females: 204-251 g
- Housing:
Before mating: 2 animals of the same sex/cage
Mating hours: 1 male and 1 female/cage
Pregnant females were housed individually
Males after mating: 2 animals/cage

- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" (ssniff Spezialdiäten GmbH, D-59494 Soest, Germany)
- Water: ad libitum, tap water
- Acclimation period: 27 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 – 70
- Air changes (per hr): above 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated in vehicle in concentrations of 10 mg/mL, 20 mg/mL and 36/50 mg/mL in the formulation laboratory of Test Facility not longer than for three days before the use. The concentration of the high dose was reduced from 50 mg/mL to 36 mg/mL according to the dose reduction on Day 6. High dose formulation was not administered on Days 4 and 5 (washout days).
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until copulation occurred.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of formulations was performed in the Analytical Laboratory of Test Facility twice during the treatment period. Five samples were taken (from different places) from each concentration (Groups 2, 3 and 4) and measured. Similarly, five samples were taken from the control solution (Group 1).
Date of sampling: December 03, 2019 and January 08, 2020;
Date of analysis: December 04, 05, 2019 and January 09, 10, 2020;
The concentration of dosing formulations was between the range of 98 and 108 % of the nominal concentrations.
Duration of treatment / exposure:
Males: 48 days (14 days pre-mating period, mating period and post-mating period)
Females: 58-65 days (depending on day of mating)
Frequency of treatment:
daily
Dose / conc.:
50 mg/kg bw/day
Remarks:
10 mg/mL
Dose / conc.:
100 mg/kg bw/day
Remarks:
20 mg/mL
Dose / conc.:
250 mg/kg bw/day
Remarks:
50 mg/mL (Dose was reduced to 180 mg/kg bw/d after deaths occurred in male animals)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting with 50, 100 and 250 mg/kg bw/day was based on results obtained in previous studies performed with Diammonium Peroxodisulphate in the rat.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (morbidity and mortality were checked twice daily)
- Cage side observations included check of skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations:
Parental males were weighed on the first day of dosing (Day 0) then weekly thereafter and on the day of necropsy (fasted body weight).
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on post-partum days 0 (within 24 hours after parturition), 4, 7, 14 and 21.

FOOD CONSUMPTION:
- Time schedule: weekly except mating phase: premating Days 7 and 13 and by weekly interval during post-mating period for male animals; premating Days 0, 7 and 13, gestation days 0, 7, 14 and 21, lactation days 0, 7, 14 and 21 for female animals.

OTHER:
DELIVERY PROCESS
- Time schedule: from gestational day 21 onwards
- Observations included: nest preparation from the bedding material and nursing of the new-borns or not. The sucking success was monitored by the presence of milk in the pups' stomach. All observations were recorded individually for each dam. The duration of gestation was recorded and was calculated from day 0 of pregnancy.
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears before the treatment started from each animal being considered for study for two weeks. Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of copulation. Vaginal smear was also prepared on the day of the necropsy.
Sperm parameters (parental animals):
Not examined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups. Particular attention was paid to the external reproductive genitals.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on Day 48
- Maternal animals: All surviving animals on post-partum days 22-24 (Days 58 - 65)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Detailed histological examinations were performed on all preserved organs and tissues of dead animals and on ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure; on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary as well as the epithelial capsule and ovarian stroma.
In addition, histological examinations were performed in organs of animals showing macroscopic findings at necropsy as follows:
- kidneys: 1/12 male and 3/12 dams in control group; 1/12 dams at 50 mg/kg bw/day; 2/12 male and 2/12 dams at 100 mg/kg bw/day; 3/10 dams at 180/250 mg/kg bw/day;
- skin: 1/12 female in control group
Organ weights: Brain weight and weight of the testes and epididymides as well as prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined.


Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 22 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination).

GROSS NECROPSY
- Gross necropsy consisted of external examinations. Particular attention was paid to the external reproductive genitals.
Statistics:
The statistical evaluation of appropriate data were performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible. Frequency of toxic response, pathological and histopathological findings by sex and dose were calculated. Results were evaluated in comparison with values of control group (i.e. control value).
Reproductive indices:
Copulatory Index, Fertility Index and Gestation Index
Offspring viability indices:
Survival Index
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Both male and female animals were normal in the control, 50 and 100 mg/kg bw/day during the entire observation period (pre-mating, mating, post-mating, gestation and lactation).
Clinical signs related to the test item were observed in male and female animals at 250 mg/kg bw/day. Signs included piloerection (8/8 male, 12/12 female), decreased activity (8/8 male, 12/12 female), decreased body tone (1/8 male, 1/12 female), diarrhea (1/8 male) and hunched back (1/8 male) between Days 1 and 5. After the dose reduction to 180 mg/kg bw/day, these animals became normal.
Mortality:
mortality observed, treatment-related
Description (incidence):
250 mg/kg bw/day dose of Diammonium Peroxodisulphate caused death of four male animals (4/12) after four-day administration on Days 4 or 5. Macroscopic and histopathological examinations revealed changes in the gastro-intestinal tract, referring to disturbances in electrolyte and water household and circulatory system along with signs of acute stress reaction in the adrenal gland and thymus due to agony.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight decreased in male animals and the body weight development was depressed in female animals at 180/250 mg/kg bw/day during the first week of the treatment (Days 0-7). After the dose reduction, the body weight gain was normalized in male and female animals but the mean body weight of male animals remained slightly below the control value up to the end of the treatment period.
Slightly reduced body weight gain at 100 mg/kg bw/day did not result in significant changes in the mean body weight during the treatment period in male animals and during the pre-mating period in female animals. In the male animals, the mean body weight and mean body weight gain were comparable in the control and 50 mg/kg bw/day during the entire observation period. In the female animals, the body weight development was comparable in the control and 50 mg/kg bw/day group during the pre-mating and gestation periods.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean daily food consumption was lowered in male and female animals at 180/250 mg/kg bw/day during the first week of the treatment (between Days 0 and 7).
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histological examination revealed lesions in the gastro-intestinal tract in dead male animals at 250 mg/kg bw/day – acute inflammation in the mucous membrane of stomach, and acute catarrh in the small and large intestines. Additionally, congestion in the liver and lungs, increased vacuolation of cortical cell in the adrenal glands and lymphocyte depletion in the spleen and thymus were also detected.
Diammonium Peroxodisulphate did not cause any toxic or other test item related lesions detectable by histological examination of the selected reproductive organs of experimental male and female animals at 180/250 mg/kg bw/day.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Serum thyroid hormones
The FT4 and TSH levels were not adversely affected in the parental male animals or in PN22 offspring at any dose levels.

Delivery data of dams
The percentage and mean number of the pre-natal (post-implantation) loss was higher in female animals at 180/250 mg/kg bw/day than in the control group and the historical control value. Although, there were no statistically significant differences between the control and 180/250 mg/kg bw/day administered group, a test item influence cannot be excluded and verified with the results of this study.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The examined parameters of the estrous cycle were not adversely by the test item (50, 100 or 180/250 mg/kg bw/day). Higher mean numbers of days of diestrous at 180/250 mg/kg bw/day might be indicative of test item influence but might also refer to the biological variation.
Reproductive performance:
no effects observed
Description (incidence and severity):
The examined parameters of reproductive performance were not adversely affected by the treatment with the test item in male and female animals at 50, 100 or 180/250 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Remarks on result:
not determinable because of methodological limitations
Remarks:
DRF
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Key result
Dose descriptor:
NOAEL
Remarks on result:
not determinable because of methodological limitations
Remarks:
DRF
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Based on the above findings the suggested dose levels for the main study (OECD 443) are as follows:
Group 1: 0 mg/kg bw/day
Group 2: 50 mg/kg bw/day
Group 3: 100 mg/kg bw/day
Group 4: 200 mg/kg bw/day (Please refer to Study No. 552-421-5133R for additional information)
Executive summary:

The subject of this study was the Pre-Test for OECD 443 Study with Diammonium Peroxodisulphate (APS) in the rat on the basis of OECD 421. The guideline is designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing. The purpose of this study was to determine the dose levels for the main study (OECD 443) and obtain information on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 50, 100 and 180/2501 mg/kg bw/day compared to control animals receiving vehicle only. Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 50, 100 and 180/250 mg/kg bw/day doses corresponding to concentrations of 0, 10, 20 and 36/50 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, distilled water. The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). The concentration of the test item in the dosing formulations used for animal’s treatment was checked two times during the study. Concentration of APS in the dosing formulations varied between the range of 98 % and 108 % of the nominal values and formulations were homogenous thereby confirming the proper dosing. All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 48 days, except for 4 dead animals). Dams were additionally exposed through the gestation period and up to lactation days 21-23 (altogether for 58 - 65 days). Non-pregnant female animals and one dam, for which no living pups remained, were administered for 48 days. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears for two weeks before the treatment (pre-experimental period) and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating and on the day of the necropsy. The dams were allowed to litter and rear their offspring up to day 21 postpartum or shortly thereafter. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 22. Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4, TSH) from 2-6 pups per litter (except for litters with 9 or less pups) on post-natal day 4, from all dams and from 2 pups per litter on post-partum/post-natal day 22 and from all parent male animals at termination. All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined.

Histopathology examination was performed on reproductive organs (testes, epididymides, prostate, and seminal vesicles with coagulating glands, uterus with oviduct, cervix, vagina and ovaries) in the control and high dose groups. Full histopathology examinations were performed on the organs and tissues of dead male animals. In addition, histopathology examinations were performed on organs showing macroscopic changes (kidneys, skin) at necropsy.

 

Results

Mortality

250 mg/kg bw/day dose of APS caused death of four male animals (4/12) after four-day administration on Days 4 or 5. Piloerection, decreased activity, decreased body tone, diarrhea and narrow eye aperture were noted for these animals during the preceding days (from Day 1 to Days 3 or 4). Changes in the gastro-intestinal tract referred to disturbances in electrolyte and water household and circulatory system along with signs of acute stress reaction in the adrenal gland and thymus due to agony.

 

Clinical observation

Test item related clinical signs were observed in male and female animals at 250 mg/kg bw/day: piloerection, decreased activity, decreased body tone, diarrhea and narrow eye aperture were observed in dead animals from Day 1 up to and including Days 3 or 4 and animals were found dead on Days 4 or 5. After the dose reduction to 180 mg/kg bw/day, these animals became normal.

 

Body weight and body weight gain

The body weight development was depressed in male and female animals at 180/250 mg/kg bw/day during the first week of the treatment (Days 0-7). After the dose reduction, the body weight gain was normalized in male and female animals but the mean body weight of male animals remained slightly below the control value up to the end of the treatment period. Slight reduction in body weight gain at 100 mg/kg bw/day did not result in significant changes in the mean body weight during the treatment period in male animals and during the pre-mating period in female animals.

 

Food consumption

The mean daily food consumption was lowered in male and female animals at 180/250 mg/kg bw/day during the first week of the treatment (between Days 0 and 7).

 

Estrous cycle

The examined parameters of the estrous cycle were not adversely by the test item (50, 100 or 180/250mg/kg bw/day). Higher mean numbers of days of diestrous at 180/250 mg/kg bw/day might be indicative of test item influence but might also refer to the biological variation.

 

Delivery data of dams

The percentage and mean number of the pre-natal (post-implantation) loss was higher in female animals at 180/250 mg/kg bw/day than in the control group and the historical control value. Although, there were no statistically significant differences between the control and 180/250 mg/kg bw/day administered group, a test item influence cannot be excluded and verified with the results of this study.

 

Reproductive performance

The examined parameters of reproductive performance were not adversely affected by the treatment with the test item in male and female animals at 50, 100 or 180/250 mg/kg bw/day. Test item influence on the slightly lower fertility index in the female animals at 180/250 mg/kg bw/day cannot be excluded entirely, although similar findings are also seen in untreated rats of this strain.

 

Serum thyroid hormones

The FT4 and TSH levels were not adversely affected in the parental male animals or in PN22 offspring at any dose levels.

 

Necropsy

Macroscopic alterations related to the effect of the test item were detected in the gastro-intestinal tract (hemorrhage, dilatation, liquid content) and on the hairs around the anus (sign of diarrhea) in dead male animals administered with 250 mg/kg bw/day at the necropsy examinations. Gross necropsy did not reveal any macroscopic changes which were related to treatment with the test item in surviving animals at 50, 100 or 180/250 mg/kg bw/day.

 

Organ weight

The weights of examined organs (absolute, relative to body and brain weights) were not adversely affected by the test item at 50, 100 or 180/250 mg/kg bw/day.

 

Histopathology

Histological examination revealed lesions in the gastro-intestinal tract in dead male animals at 250 mg/kg bw/day – acute inflammation in the mucous membrane of stomach, and acute catarrh in the small and large intestines. Additionally, congestion in the liver and lungs, increased vacuolation of cortical cell in the adrenal glands and lymphocyte depletion in the spleen and thymus were also detected. APS did not cause any toxic or other test item related lesions detectable by histological examination of the selected reproductive organs of experimental male and female animals at 180/250 mg/kg bw/day.

 

Offspring

The offspring’s development was not adversely influenced at any dose level. No effects on the mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected.

 

Conclusion

Under the conditions of the present study, APS caused death (male), clinical signs (male and female), depressed body weight development and food consumption (male and female), macroscopic and histopathology changes in stomach and intestines (male) in Han:WIST rats at 250 mg/kg bw/day by oral gavage as investigated in this study. After the dose reduction to 180 mg/kg bw/day, clinical signs ceased after some days and there were no adverse effects in the examined parameters. Nonetheless the body weight of the males had not recovered by the end of the study. There were no test item related adverse effects at 50 or 100 mg/kg bw/day.

APS did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female animals at 50, 100 or 180/250 mg/kg bw/day.

The development of the F1 offspring was not impaired from birth to post-natal day 21 after repeated oral administration of dams at 50, 100 or 180/250 mg/kg bw/day.

The suggested dose levels for the main study (OECD 443) are as follows:

Group 1: 0 mg/kg bw/day

Group 2: 50 mg/kg bw/day

Group 3: 100 mg/kg bw/day

Group 4: 200 mg/kg bw/day (Please refer to Study No. 552-421-5133R for additional information)

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
DRF for OECD 443
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2020-04-21 to 2020-05-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
no guideline followed
Principles of method if other than guideline:
In the DRF for OECD 443 effects at 180 mg/kg bw/d were considered as not severe enough to define it as a high dose in the main test. Thus, an additional 14 day DRF with 200 mg/kg bw/d was performed.
GLP compliance:
no
Remarks:
This study was not performed according to GLP compliances, however, the principles of GLP were followed and all data were recorded and retained.
Limit test:
yes
Species:
rat
Strain:
Wistar
Route of administration:
oral: gavage
Vehicle:
water
Duration of treatment / exposure:
14 days
Frequency of treatment:
daily
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Effects at 180 mg/kg bw/d in the DRF for the OECD 443 were not severe enough. Thus 200 mg/kg bw/d was tested additionally.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Detailed observation, once daily; Mortality/ morbidity: twice a day throughout the study

BODY WEIGHT: Yes
- Time schedule for examinations: Just before the first administration and on Days 0, 3, 7, 10 and 13.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: on Days 0, 7, and 13
Postmortem examinations (parental animals):
SACRIFICE
- all animals: on study day 14

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including gastro-intestinal tract, eyes and mucous membranes, that were observed with particular attention.
Statistics:
Statistical analysis was done with SPSS PC+ software package for the body weight, body weight gain and food consumption data.
Frequency of clinical signs and pathological findings by sex and dose was calculated.
Clinical signs:
no effects observed
Description (incidence and severity):
The general physical condition and behavior of animals were considered to be normal throughout the entire observation period. No clinical signs were detected in the animals (male and female) in the control or 200 mg/kg bw/day groups at the daily observations.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight development was not adversely affected by the treatment with 200 mg/kg bw/day throughout the 14-day treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean daily food consumption was not adversely affected at 200 mg/kg bw/day (male and female) during the 14-Day observation period.
Key result
Dose descriptor:
NOAEL
Remarks on result:
not determinable because of methodological limitations
Remarks:
DRF
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Remarks on result:
not determinable because of methodological limitations
Remarks:
F1 not included in this DRF
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Conclusions:
Diammonium Peroxodisulphate caused no signs of toxicity (clinical signs, body weight and body weight gain, food consumption, necropsy findings) in male or female Han:WIST rats after the consecutive 14-day oral (by gavage) administration of 200 mg/kg bw/day.
Executive summary:

The purpose of this study was to obtain information on the effect of the test item when administered by gavage to male and female Han: WIST rats at 200 mg/kg bw/day dose for 14 days compared to control animals receiving vehicle (distilled water) only and to determine the highest dose level for the main study (OECD 443).

The dose setting of 200 mg/kg bw/day was based on the results of previously performed study with the test item “Pre-Test for OECD 443 Study with Diammonium Peroxodisulphate in the Rat” (Study no. 552-421-5133).

The test item was administered by gavage to Han:WIST rats (n=6 animals/ sex/ group) once a day at 0 (vehicle control) and 200 mg/kg bw/day corresponding to concentrations of 0 and 40 mg/mL for 14 days. The application volume was 5 mL/kg bw.

Detailed clinical observations were made on all animals once a day, after treatment at approximately the same time. Body weight was determined twice a week and food consumption was measured weekly. Gross pathology examinations were conducted one day after the last administration (Day 14).

The results of this study were summarised as follows:

No animal died during the study.

The control (6/6 male and 6/6 female) and test item treated animals (5/6, male and 6/6 female) showed no clinical signs during the treatment period. Diarrhea was noted for one male animal (1/6) transiently on Day 0. The behavior and the general state of the test animals were considered to be normal during the study.

The mean body weight, body weight gain and the mean daily food consumption were comparable to their control in male and female animals at 200 mg/kg bw/day during the 14-day observation period.

Specific macroscopic alterations related to treatment with the test item were not observed during the terminal necropsy of treated animals.

Conclusion

Diammonium Peroxodisulphate caused no signs of toxicity (clinical signs, body weight and body weight gain, food consumption, necropsy observations) in male or female Han:WIST rats after the consecutive 14-day oral (by gavage) administration of 200 mg/kg bw/day. Thus 200 mg/kg bw/day was used as highest dose in the main study of OECD 443.

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-01-30 to 2004-12-13
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD (SD)IGS BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories; Raleigh; North Caroline (USA)
- Age at study initiation: (P) 11 wks
- Weight at study initiation: (P) Males: 359 - 436 g; Females: 212 - 280 g
- Fasting period before study: no
- Housing: Males and females were housed individually (except during mating)
- Diet: Powdered PMI Certified Rodent Diet #5002 (PMI Feeds, Inc.; St Louis, Missouri) ad libitum
- Water: ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature: 18 - 26 °C
- Humidity: 30 - 70 % R.H.
- Air changes: a minimum of 10 air changes per hour
- Photoperiod: 12 hrs dark/12 hrs light
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Study design:
Male and female Crl:CD (SD)IGS BR rats were assigned to 4 groups (12/sex/group). Each group received a control diet consisting of powdered PMI Certified Rodent diet #5002 (PMI Feeds Inc., St. Louis, Missouri) or the same diet containing diammonium persulfate at concentrations designed to provide target doses of 40, 100 250 mg/kg/day based on mean weekly body weight and mean weekly food consumption data for each sex at each dose level.
Dose administration:
Males were administered with test material in the diet for three weeks prior to mating, during the mating period (four days) and approximately five weeks after mating. Females were dosed throughout the 3-week pre-mating period, during the 4-day mating period, throughout gestation (22 days) and during the first four days of lactation. Depending on what day the female was confirmed pregnant, the total days of dose administration varied from 47 to 51 days. The animals were dosed until the day of sacrifice (prior to removal from the room).
Details on mating procedure:
Breeding:
Animals from respective groups were mated by placing one female in the breeding cage of a male from the same dose group. A record of mating pairs was maintained. Once mating occurred, the males and females were separated. The maximum mating period was 14 days. Females for which no evidence of mating was detected after a total of 14 days were placed in nesting boxes.
Confirmation of mating:
During mating, a daily inspection was be made for the presence of a retained copulatory plug, multiple copulatory plugs on the cage tray, or vaginal sperm. The day sperm or a plug was observed was designed to be day 0 of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration of diammonium persulfate in rodent feed samples were determined using the measurement of the exothermic enthalpy delta H (J/g) by DSC of the test samples and comparison with a standard calibration curve. The method was evaluated and validated and found to be suitable for the analytical determination of diammonium persulfate in rodent diet.

Experimental procedures:
Sample preparation:
About 1 g to 10 g of sample was taken with a spatula from the container and ground for 3 minutes in a cleaned mortar and pestle. About 10 - 30 mg of the ground sample was transferred into a tared round DSC sample pan. By using a small steel cylinder, which was the same size as the pan, the powdery sample in the pan was compressed. The sample was weighed to ± 0.01 mg with an Autobalance AD 6. The weight of the sample was recorded on a sample weighing sheet. The sample pan containing the sample was then transferred into the sample holder of the DSC system for analysis immediately.
Instrumentation and Analysis:
DSC System:
Perkin Elmer Pyris 1 Differential Scanning Calorimeter; Serial Number: S73N0061703;
Balance:
PE AD 6 Autobalance; Balance Serial #: 655C04;
Sample Analysis:
DSC Mode used: Ice Bath;
Sample atmosphere: Nitrogen, 20 mL/min;
Sample pan: Aluminium, open pan;
Temperature Program: Hold at 30 °C for 1 minute; Heat from 30 °C to 220 °C with a rate of 10 °C/min;
Calculation:
Exothermic enthalpy delta H (J/g) of each sample in temperature range between 160 °C to 210 °C was calculated with Pyris Software provided by the DSC system.
A five point standard curve of ammonium persulfate concentration (ppm) versus delta H (J/g) was developed in a separate method development/validation procedure. The following equation, which was obtained with the five point standard curve, was used to calculate the ammonium persulfate concentration of each sample:
Concentration (ppm) = (delta H + 0.0052671)/0.0007350
where:
0.0052671 = Intercept of the standard curve
0.0007350 = Slope of the standard curve
Duration of treatment / exposure:
Not applicable
Frequency of treatment:
Not applicable
Details on study schedule:
The purpose of the study was to provide information regarding possible effects on mating, reproduction, and/or development in male and female rats. Males were administered with test substance in the diet three weeks prior to mating, during the mating period (four days) and approximately five weeks after mating.
Females were dosed throughout the 3-week pre-mating period, during the 4-day mating period, throughout gestation (22 days) and during the first four days of lactation. The total length of exposure was approximately 51 days for females (depending on when the female was confirmed pregnant) and 56 days for males.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
in diet
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
in diet
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
in diet
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
in diet
No. of animals per sex per dose:
12 animals per sex and per dose group
Control animals:
yes, plain diet
Details on study design:
Dietary formulations:
Diets were mixed at least weekly according to the mixing procedure approved by the sponsor. For each high-dose formulation prepared (whether for one or both sexes, depending on the phase of the study), the total amount of test substance needed for that high-dose stock formulation was weighed and the finely ground with a mortar and pestle. The finely ground test article was gradually mixed into approximately 200 g of the PMI powdered diet. This preliminary premixture of test article and powdered diet was then placed in a Hobart mixer, and the remaining powdered diet needed to achieve the high-dose stock formulation was gradually added to the test article and feed mixture. After completion of the additional powdered diet, the high dose stock formulation was mixed for at least 30 minutes. Once this mix was completed, the low and mid levels were prepared by taking the appropriate amount of the high-dose stock formulation and diluting it to the appropriate dietary concentration needed for each dose level by gradually adding powdered feed to achieve the correct precalculated weight. Calculated diet concentrations were based on historical body weight and food consumption data for Days 0 to 7 and based on current food consumption and body weight data for the appropriate sex after Day 7. All diet formulations were stored at room temperature until dosed. Dose concentrations were based on test article as supplied (i.e., no correction factor was used when calculating the amount of test article to be use).
Positive control:
Not indicated
Parental animals: Observations and examinations:
Assessment of toxicity was based on mortality, clinical observations, body weights and weight change (during premating and mating for both sexes, during gestation and lactation for females, and postmating for males), food consumption (during premating for both sexes, during gestation and lactation for females, and postmating for males), pregnancy rates and maternal data, litter data (litter size, sex and weight of pups, live and dead pups), anatomic pathology, organ weights, histopathology of selected tissues, and staging of spermatogenesis.
Oestrous cyclicity (parental animals):
Not indicated
Sperm parameters (parental animals):
The following parameters were examined:
- Normal progression of spermatogenesis at necropsy.
- Stages of the spermatogenic cycle.
- Testicles examination.
Litter observations:
Litter size, sex and weight of pups, live and dead pups were examined.
Postmortem examinations (parental animals):
The necropsy included an examination of the external features of the carcass, external body orifices, the abdominal, thoracic, and cranial cavities, and organs/tissues.
At scheduled necropsy, the following organs were weighed: brain; liver; kidneys; prostate with seminal vesicles including secretions; testes, epididymides; ovaries; uterus (with cervix).
The following tissues from adult animals were preserved: cervix, caecum, coagulating gland, colon, duodenum; epididymides, oesophagus, ileum, jejunum, lesions, ovary with oviducts, prostate, rectum, seminal vesicle, stomach, testis, uterus, vagina.
Postmortem examinations (offspring):
At Day 4: Litter size and sex, weight, and abnormal observations of individual offspring were recorded. All dead pups were examined for cervical, thoracic and abdominal viscera abnormities and preserved in alcohol. Live pups were sacrificed, examined for gross external abnormalities, and discarded without necropsy.
Statistics:
Appropriate maternal and litter data were analyzed using analysis of variance (ANOVA) techniques; Levene's test was employed to analyze homogeneity of variances and Dunnett's t-test served as the post-hoc group comparison test. Calculated indices were analyzed using Fisher-Irwin exact test. Pup weights were analyzed by analysis of covariance (ANCOVA) techniques using the litter size as the covariate. Group comparisons were evaluated at the 5 % and 1 % two-tailed probability levels for all statistic analyses; a one-tailed analysis was used to determine significance for trend where appropriate.
Reproductive indices:
Fertility indices:
Female fertility = (number of females pregnant/total number females mated) x 100
Male fertility = (no. of males shown to be fertile/total number of males mated) x 100
Gestation index = (number of live litters born/number of pregnancies) x 100.
Offspring viability indices:
Viability index = (number of pups surviving to Day 4/number of pups born alive) x 100
Percent males (Days 0 and 4) = (number of males/total number of pups) x 100.
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
not specified
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
There was no mortality. All animals survived to terminal sacrifice with the exception of two non pregnant females dosed at 100 and 250 mg/kg/day, respectively, which were sacrificed on day 26 due to their failure to deliver.
There were no treatment-related clinical signs of toxicity observed in F0 parents of either sex or in F1 pups at any treatment level. Remarkable clinical signs in the F0 parents and F1 pups were not attributed to treatment with diammonium persulfate, as they occurred sporadically, were of short duration, and did not demonstrate a dose response.
Normal progression of spermatogenesis was observed in male parental animals at necropsy. All stages of the spermatogenic cycle were present. In the testicles examination there was no indication of any test substance effects.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: male and female toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: male and female fertility performance
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
There were no treatment-related clinical signs of toxicity observed in F1 pups at any treatment level. Remarkable clinical signs in the F1 pups were not attributed to treatment with diammonium persulfate, as they occurred sporadically, were of short duration, and did not demonstrate a dose response.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 250 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: pup viability and development
Key result
Reproductive effects observed:
not specified
Conclusions:
The NOAEL for male and female toxicity, for male and female fertility performance and for embryo/foetal viability and development was >= 250 mg/kg/day.
Executive summary:

Diammonium persulfate was tested for oral reproductive/developmental toxicity in a screening test with rats according to OECD guideline 421.The purpose of this study was to obtain initial information on the possible effects of the test item on reproduction and development when administered orally in the diet to Crl:CD (SD)IGS BR rats at doses of 40, 100 and 250 mg/kg bw/day compared to control animals (plain diet only).

There were no treatment-related clinical signs of toxicity observed in F0 parents of either sex or in F1 pups at any treatment level. Remarkable clinical signs in the F0 parents and F1 pups were not attributed to treatment with diammonium persulfate, as they occurred sporadically, were of short duration, and did not demonstrate a dose response. No significant changes were observed in male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and in development of the F1 offspring from conception to day 4 postpartum. In conclusion, under the conditions of this study, the NOAEL for male and female toxicity, the NOAEL for male and female fertility performance and the NOAEL for F1 viability and development was >= 250 mg/kg/day.

Endpoint:
one-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
other: APS
Sex:
female
Basis for effect level:
reproductive performance
Remarks on result:
other: Only treatment related effects secondary to systemic effects. Lower incidence of dams delivered at 100 and 200/180 mg/kg bw/day was related to the premature death around the day of birth.
Key result
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day
Based on:
other: APS
Sex:
male
Basis for effect level:
other: No effects observed regarding male reproductive performance up to the highest dose tested.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
other: APS
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Based on:
other: APS
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: Cohort 1A and 1B
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day
Based on:
other: APS
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: reduced development (surface righting reflex, pinna detachment, eye opening)
Remarks on result:
other: Offspring
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Endpoint:
one-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Remarks on result:
not determinable because of methodological limitations
Remarks:
DRF
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Remarks on result:
not determinable because of methodological limitations
Remarks:
DRF
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Endpoint:
one-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Remarks on result:
not determinable because of methodological limitations
Remarks:
DRF
Critical effects observed:
not specified
Key result
Dose descriptor:
NOAEL
Remarks on result:
not determinable because of methodological limitations
Remarks:
F1 not included in this DRF
Critical effects observed:
not specified
Key result
Reproductive effects observed:
not specified
Endpoint:
one-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: male and female toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: male and female fertility performance
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 250 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: pup viability and development
Key result
Reproductive effects observed:
not specified
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-06-23 to 2021-05-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
June 25, 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals
: 10 weeks
- Basis for dose level selection: DRF (on the basis of OECD 421) was performed (rf. to study entry for study number 552-421-5133 and 552-421-5133R)
- Inclusion/exclusion of extension of Cohort 1B: not triggered by available data with the test item
- Termination time for F2
: not applicable
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B
: not triggered by available data with the test item
- Inclusion/exclusion of developmental immunotoxicity Cohort 3
: not triggered by available data with the test item
- Route of administration
: gavage; A 14 d palatability pre-test has been performed with APS in diet at doses of 250 and 500 mg/kg bw/d (please refer to IUCLID section 7.5.1). The test substance proved to be not stable in diet (analytical study not reported in the dossier).
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in reproduction toxicity studies and well-known fertility parameters.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:
Toxi-Coop Zrt., Cserkesz u. 90., 1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 8 wks
- Weight at study initiation: (P) Males: 237 – 296 g; Females: 166 – 205 g;
- Housing:
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female/cage
Mated females: individually
Males after mating: 2 animals/cage
F1 offspring (after weaning): 2 or three animals of the same sex/cage
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" (ssniff Spezialdiäten GmbH, D-59494 Soest, Germany)
- Water: ad libitum, tap water
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle. Formulations were prepared in the formulation laboratory of the Test Facility and were used within five days. Formulations were stored at room temperature.

VEHICLE
- Concentration in vehicle: 10, 20 and 40/36 mg/mL
- Treatment Volume: A constant treatment volume of 5 mL dose preparation/kg body weight was administered in all groups.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility if less than 20 pregnant females per group have been achieved.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: none
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of formulations was performed in the Analytical Laboratory of Test Facility. Aqueous formulation samples were diluted with ultra-pure water
and then analysed by an LC/MS/MS method.
Five samples were taken from different places from each concentration (Groups 2, 3 and 4) and measured on five occasions. Similarly, five samples were taken from the control solution (Group 1) from different places and analyzed.
Sampling date: July 07, 2021; September 28, 2021; November 24, 2021; December 16, 2021; January 06, 2021;
Analysis date: July 08-09, 2021; September 28, 2021; November 27, 2021; December 16, 2021; January 06-07, 2021;
Samples were stored at room temperature until analysis.
(i) Stability analysis:
Stock Solution Stability (5 ± 3°C) = 1 day
Stability in the Autosampler = at least 33 hours
Stability in distilled water (RT, refrigerated) = 5 days (1 and 50 mg/mL)
(ii) Homogeneity analysis: The formulations proved to be homogeneous. Deviation in the concentration of the replicate samples was not more than 8%.
(iii)The Diammonium Peroxodisulphate (APS) concentrations in the samples varied in the range from 93% to 106 % in comparison to the nominal values.
Duration of treatment / exposure:
(P) Males: 71-73 days before mating and up to 14 days mating and until weaning of offspring
(P) Females: 71-73 days before mating, up to 14 days and throughout pregnancy and at least up to and including post-partum day 21 or up to the day before sacrifice
(approx. 18 wks)
(F1A) Males: 104-109 days at weaning, during growth into adulthood
(F1A) Females: 104-109 days at weaning, during growth into adulthood
(approx. 12 wks)
(F1B) Males: 110-115 days at weaning, during growth into adulthood
(F1B) Females: 110-115 days at weaning, during growth into adulthood
(approx. 13 wks)
Frequency of treatment:
daily, 7 days each week
Details on study schedule:
- Age at mating of the mated animals in the study: 19 weeks
Dose / conc.:
50 mg/kg bw/day
Remarks:
10 mg/mL
Dose / conc.:
100 mg/kg bw/day
Remarks:
20 mg/mL
Dose / conc.:
200 mg/kg bw/day
Remarks:
40/36 mg/mL (dose was reduced back to 180 mg/kg bw/d due to moribund condition of male and female animals)
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose setting is based on findings obtained from previous studies with Diammonium Peroxodisulphate in rats (Pre-Tests for OECD 443 Study with Diammonium Peroxodisulphate in the Rat, study nos. 552-421-5133 and 552-421-5133R) and in agreement with the Sponsor. Both studies are included in the IUCLID under section 7.8.1. The dose range finder (study no. 552-421-5133) was performed on the basis of OECD TG 421 at 50, 100 and 250 mg/kg bw/day. The high dose was lowered after 4 days of treatment due to mortality of four male animals. The dose reduction to 180 mg/kg bw/day led to ceasing of clinical signs after some days. No adverse effects were observed in the examined parameters. Only the body weight development of male animals had not recovered by the end of the study. Severe effects were fastly seen after 4 days of treatment. In order to test a high dose in the main study with the aim to induce toxic effects but no mortality another 14-day study was performed at 200 mg/kg bw/day (study no. 552-421-5133R). The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. In case of severe signs of toxicity, the high dose will be reduced in the course of the study.
- Fasting period before blood sampling for clinical biochemistry: yes, 16 hours
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: same as weighing

BODY WEIGHT: Yes
- Time schedule for examinations:
Parental males were weighed on the first day of dosing (day 0) and weekly thereafter.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 2, 4, 7, 9, 11, 14, 16, 18, 21 and on post-partum days 0 (within 24 hours after parturition), 4, 7, 14 and 21. Body weight of the pregnant female animals was only evaluated statistically on gestation days 0, 7, 14 and 21. Body weight data was reported individually for adult animals.

F1 animals selected for follow-up examinations were weighed on post-natal day 22, then twice a week during the two weeks following weaning, and once weekly thereafter.
For selected F1 offspring, the body weight was recorded on the day when they attain puberty (completion of balano-preputial separation or vaginal patency).

Fasted body weight was measured on the day of necropsy for all animals (P and F1).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears from each parental (P) female animal daily for two weeks before the mating started (i.e., after 8 weeks of pre-mating treatment) and daily for two weeks before the necropsy for F1 Cohort 1A and Cohort 1B female animals. Vaginal smears were also prepared and estrous cycle was monitored daily during the mating period until evidence of copulation (P) and on the day of the necropsy of all female animals (P and F1 Cohort 1A and Cohort 1B). Vaginal smears were examined for all F1 Cohort 1A females selected for follow-up examinations after the onset of vaginal patency until the first cornified smear was recorded thus determining the time interval between these events.
Vaginal smears were stained with 1 % aqueous methylene blue solution. After drying, the smears were examined with a light microscope.
Sperm parameters (parental animals):
Sperm parameters were measured in all control and high dose male animals in P generation and in F1 generation in Cohort 1A.
The one-side testes and epididymides were used for examinations. The weights of one-side testes and epididymides were determined and recorded.
Sperm from the ductus deferens was collected for evaluation of sperm motility and morphology at the necropsy. Both numbers of motile and immotile sperms were recorded. Two samples were prepared from each animal. For the determination of the sperm motility, the mean percentage of motile sperms was determined.
A morphological evaluation of ductus deferens sperms sample was performed from the same animals. Sperm was examined as fixed, wet preparations and classified as either normal or abnormal (isolated heads, misshapen heads and/or tails).
The epididymis was used for enumeration of cauda epididymis sperm reserves. The total number of sperms in homogenization was enumerated. The testis and epididymidis were frozen and enumeration was performed later.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other. Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; possible cause of death was determined for pups born or found dead, if it can be identified.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [after the optionally extended post-mating period]
- Maternal animals: All surviving animals [on post-partum day 22 or shortly after]

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
All organs showing macroscopic lesions and the following organs were preserved for all parental (P) and Cohort 1A and Cohort 1B animals (male and female):

- Adrenal glands
- Bone with marrow and joint (femur)
- Brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata)
- Eyes (lachrymal gland with Harderian glands and optic nerve)
- Mammary gland (male and female)
- Heart
- Kidneys
- Large intestines (caecum, colon, rectum)
- Liver
- Lungs (with main stem bronchi; inflation with fixative and then immersion;)
- Lymph nodes (submandibular, mesenteric; popliteal for Cohort 1 animals)
- Muscle (quadriceps)
- Esophagus
- Pancreas
- Pituitary
- Salivary glands (submandibular)
- Sciatic nerve
- Sexual organs (testes, epididymides, seminal vesicle with coagulating gland, prostate ovaries, uterus with oviduct, vagina)
- Small intestines (duodenum, ileum, jejunum including Peyer’s patches)
- Spinal cord (at three levels: cervical, mid-thoracic and lumbar)
- Spleen
- Stomach
- Thymus
- Thyroid + Parathyroid
- Trachea
- Urinary bladder

The wet weight of the following organs were determined from all parental animals and all adult F1 animals of Cohort 1A:
- uterus (with oviducts and cervix)
- ovaries
- testes
- epididymides
- prostate (dorsolateral and ventral parts combined)
- seminal vesicles with coagulating glands as one unit (with their fluids)
- brain,
- liver,
- kidneys,
- spleen,
- thymus,
- pituitary,
- thyroid glands (post-fixation)
- adrenal glands

In animals of Cohort 1B, the weight of following organs was determined:
- uterus (with oviducts and cervix)
- ovaries
- testes
- epididymides
- prostate (dorsolateral and ventral parts combined)
- seminal vesicles with coagulating glands as one unit (with their fluids)
- brain
- pituitary
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 22 days of age
- These animals were subjected to postmortem examinations macroscopic and microscopic examination as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated above were prepared for microscopic examination and weighed, respectively.

IMMUNOTOXIC EXAMINATION

At termination, weighing of the lymph nodes and splenic lymphocyte subpopulation analysis will be performed in 10 male and 10 female Cohort 1A animals from each group (1 male or 1 female per litter; all litters represented by at least 1 pup; randomly selected) as follows:
- weighing of the lymph nodes associated with and distant from the route of exposure (submandibular and popliteal lymph nodes);
- splenic lymphocyte subpopulation analysis (CD4+ and CD8+ Tlymphocytes, B lymphocytes and natural killer cells) by flow cytometry method using one half of the spleen
Statistics:
The statistical evaluation of appropriate data (marked above with †) was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant results at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose were calculated. Results were evaluated in comparison with values of control group (i.e. control value).
Reproductive indices:
Copulatory Index (Measure of animals ability to mate):
Males: Number of males with confirmed mating / Total number of males cohabited x 100
Females: Number of sperm positive females / Total number of females cohabited x 100

Fertility Index (Measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant):
Males: Number of males impregnating a females / Total number of males with confirmed mating x 100
Females: Number of pregnant females / Number of sperm positive females x 100

Gestation Index (Measure of pregnancy that provides at least one live pup):
Number of females with live born pups / Number of pregnant females x 100
Offspring viability indices:
Post-implantation mortality: Number of implantations – Number of liveborns / Number of implantation x 100

Post-natal mortality: Number of liveborns – Number of live pups on PND 13 / Number of liveborns x 100

Survival Index: Number of live pups on PND 13 / Number of liveborns x 100

Sex ratio: Number of pups examined – Number of pups males (females) / Number of pups examined x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Signs of systemic toxicity related to the test item treatment were detected in pregnant female animals at 100 mg/kg bw/day and in male and female animals at 200/180 mg/kg bw/day during the course of daily clinical observations.
The main clinical signs appeared at two peak-periods in male animals at 200/180 mg/kg bw/day: decreased activity, decreased body tone, diarrhea, piloerection or sanguineous nasal orifice between Days 4 and 10 and decreased activity and decreased body tone in some animals from Day 116 up to and including Day 124.
In female animals at 100 and 200/180 mg/kg bw/day, the main signs cumulated around the delivery. Decreased activity, piloerection, decreased body tone, diarrhea, black discharge around eyes, sanguineous nasal orifice, and sanguineous fore limbs were detected during the last few days of gestation (G17-21) and during the first two days of lactation (L0-L1).
Signs of systemic toxicity related to the test item treatment were detected in female animals at 100 mg/kg bw/day and in male and female animals at 200/180 mg/kg bw/day during the course of weekly clinical observations. For further details please refer to "Any other information on results incl. tables".
Mortality:
mortality observed, treatment-related
Description (incidence):
Test item related mortality was observed in pregnant female animals at 100 and 200/180 mg/kg bw/day.
One pregnant female animal at 100 mg/kg bw/day (1/24) and 6 pregnant female animals at 200/180 mg/kg bw/day (6/24) were found dead close to delivery. Additionally, one male animal in the control group (1/24) and 4 pregnant female animals at 200/180 mg/kg bw/day (4/24) were in moribund state and were early euthanized.
Based on necropsy and histopathological findings, the collapse of fluid balance caused the death/ moribund state of these pregnant female animals.
The male animal in the control group presumably died in consequence of an individual lesion.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight development was reduced in parental male animals administered with 100 and 200/180 mg/kg bw/day during the entire treatment period and in female animals at 200/180 mg/kg bw/day during gestation period.
Slight changes in the mean body weight and mean body weight gain of male animals at 50 mg/kg bw/day during the entire treatment period and in female animals at 100 mg/kg bw/day during the gestation period were judged to be toxicologically irrelevant as the difference with respect to the control remained less than 10 % both in male and female animals.
The mean body weight was lower than in the control group in male animals in all test item administered groups reaching statistical significances in several cases at 50 mg/kg bw/day (Days 49, 56, 77, 91 and between Days 105 and 132), at 100 mg/kg bw/day (on Day 7, between Days 35-70 and 77-132) and at 200/180 mg/kg bw/day (from Day 7 each week up to the termination on Day 132).
The mean body weight gain of male animals, remained below the control vale in the most cases and the difference with respect to the control was statistically significant on several days as follows:
- at 50 mg/kg bw/day: between Days 0-7, 21-28, 42-49, 105-112, 112-119; between Days 0 and 132;
- at 100 mg/kg bw/day: between Days 0-7, 28-35, 35-42, by weekly interval between Days 91-119; between Days 0 and 132;
- at 200/180 mg/kg bw/day: Days 0-7, 21-49 by weekly interval, between Days 91-119 by weekly interval; between Days 0 and 132;
The summarized body weight gain was dose dependently lower than in the control group at 50, 100 and 200/180 mg/kg bw/day (-13, -19 and -41 %, respectively).
In the female animals, the mean body weight was comparable to the control at 50, 100 and 200/180 mg/kg bw/day during the pre-mating period. Some sporadic statistically significant difference with respect to the control were detected at lower mean body weight gain at 100 and 200/180 mg/kg bw/day between Days 0 and 7 and at the higher mean body weight gain at 50, 100 and 200/180 mg/kg bw/day between Days 14 and 21 as well as at 200/180 mg/kg bw/day between Days 42-49. The summarized body weight gain was lower than in the control at 100 mg/kg bw/day. However, these minor differences in the mean body weight gain were not dose related and had no influence on the mean body weight of female animals. Therefore, these differences were considered to be of no toxicological relevance.
The mean body weight and body weight gain was comparable in the control and test item treated female animals at 50 mg/kg bw/day during the gestation and lactation periods. Statistical significances with respect to the control were detected at the lower mean body weight in female animals at 100 and 200/180 mg/kg bw/day on gestation Day 21 as well as on lactation days 0, 4, 7 and 14.
Statistical significance with respect to the control was noted for the slightly lower mean body weight gain of dams at 100 and 200/180 mg/kg bw/day between gestation days 14-21 and when summarized between between gestation days 0 and 21. During the lactation period, the mean body weight gain exceeded the control in dams at 100 and 200/180 mg/kg bw/day between lactation days 14 and 21 and at 200/180 mg/kg bw/day if summarized between lactation days 0 and 21.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistical significance with respect to the control was detected at the lower mean daily food consumption of parental male animals at 50 mg/kg bw/day between Days 112-119, at 100 mg/kg bw/day between Days 0-7, 63-70, 112-119, 119-126 and at 200/180 mg/kg bw/day between Days 0-7, 21-28, 28-35, by weekly interval between Days 42-70, between Days 77-84, by weekly interval between Days 91-119 and between Days 126-132.
The mean daily food consumption was similar to the control in female animal at 50 mg/kg bw/day during the pre-mating, gestation and lactation period except for lactation days 0-4, when the mean daily food consumption significantly exceeded the control (+13 %).
In the parental female animals, the mean daily food consumption was statistically significantly lower at 100 and 200/180 mg/kg bw/day between Days 0-7 comparing to the control. Sporadic statistically significant differences with respect to the control were detected independently from doses at 100 mg/kg bw/day between Days 7-14, 28-35, 35-42, 49-56, 56-63 and at 200/180 mg/kg bw/day between Days 49-56 during the pre-mating period.
The mean daily food consumption was lower than in the control at 100 mg/kg bw/day during the entire gestation period (G0-G21 by weekly interval, up to 10 %) and at 200/180 mg/kg bw/day between gestation days 14-21 (-25 %).
During the lactation period, significantly lower mean daily food consumption was observed at 100 mg/kg bw/day between lactation days 7-14 and 14-21 (21%) and at 200/180 mg/kg bw/day between lactation days 0-4 (20%), 7-14 (29 %) and 14-21 (30 %).
The above food consumption decreased dose dependently and is related to treatment. The lower food consumption is also in compliance with the negative body weight development in both sexes excluding the lactation period in female animals of 100 and 200/180 mg/kg bw/day. Here a significantly higher body weight gain was observed compared to control.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no test item related adverse changes in the examined hematological parameters in parental male or female animals at 50, 100 or 200/180 mg/kg bw/day.
In the male animals, statistical significance was detected at the slightly shorter mean prothrombin time (PT) at 50 mg/kg bw/day when compared to the control.
At 100 mg/kg bw/day, shorter mean prothrombin time and activated partial thromboplastin time (APTT) and elevated platelet count (PLT) were observed in male animals comparing to the control.
Statistical significance with respect to the control was detected at the slightly higher mean percentage of neutrophil granulocytes (NEU) along with lowered mean percentage of lymphocytes (LYM), at the shorter mean prothrombin time and activated partial thromboplastin time in male animals at 200/180 mg/kg bw/day.
In the female animals at 50 mg/kg bw/day, all the examined hematological and blood coagulation parameters were comparable to the control.
Statistical significance was detected at the slightly higher mean hematocrit value (HCT), lower mean corpuscular hemoglobin content (MCH) and at the shorter mean activated partial thromboplastin time in female animals at 100 mg/kg bw/day when compared to the control.
At 200/180 mg/kg bw/day, significantly lower mean hemoglobin content (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin content and shorter mean prothrombin time and activated partial thromboplastin time were observed in female animals.
The individual values HGB, HCT, MCV, MCH, PT, APTT and PLT met well the historical control (i.e., values were within or close to the ranges) in male and female animals, where relevant. There were no changes in related hematological parameters. Therefore, the differences in these parameters were considered to have little or no toxicological relevance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The examined clinical chemistry parameters were not adversely affected in parental male or female animals at 50, 100 or 200/180 mg/kg bw/day.
An elevation of mean activity of alanine aminotransferase (ALT) at 100 mg/kg bw/day (male) and 200/180 mg/kg bw/day (male and female) and lowered albumin (ALB) and total protein concentration (TPROT) at 100 or 200/180 mg/kg bw/day (male or female) might be indicative of changed renal function related to the altered demand.
In the male animals, clinical chemistry investigations revealed a slightly but statistically significantly lower mean concentration of total protein at 50 mg/kg bw/day. TPROT value was within the historical control. A significantly higher mean activity of alanine aminotransferase,. lowered mean concentration of albumin (ALB) and total protein was observed at 100 mg/kg bw/day that were within the historical control., At 200/180 mg/kg bw/day a statistically significantly higher mean activity of alanine aminotransferase (ALT), lowered mean concentration of creatinine (CREA), glucose (GLUC), albumin and total protein were measured.
In the female animals, a statistically significant difference with respect to the control was detected at the lower mean concentration of potassium (K+) at 50 mg/kg bw/day, at the lower mean concentration of glucose at 100 mg/kg bw/day, as well as at the higher mean activity of alanine aminotransferase, lowered mean concentration of glucose and albumin at 200/180 mg/kg bw/day. The lower glucose value in the highest dose was outside the historical control.
The changes in ALT, CREA, GLUC, K+, ALB and TPROT were judged to be related to presumed increased metabolic and excretory activity. Toxicological relevance of these changes is questionable due to the minor degree (values were within normal ranges) or the lack of related findings (clinical and organ pathology).
Endocrine findings:
not examined
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
There were no test item related adverse changes in the examined urine parameters in parental male or female animals at 50, 100 or 200/180 mg/kg bw/day.
The examined urine parameters were comparable in the parental male animals in the control and 50 mg/kg bw/day and in female animals in the control, 50 and 100 mg/kg bw/day groups.
Urinalysis revealed statistically significantly lower mean pH (-34 %) of the urine in male animals at 100 and 200/180 mg/kg bw/day and in female animals at 200/180 mg/kg bw/day (-32 %) as well as slightly elevated specific gravity (< 1 %) in female animals at 200/180 mg/kg bw/day in comparison with their control.
These minor changes were considered to be toxicologically not relevant as there were no related renal findings.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic for the sexually mature organism in all parental male animals in the control and 200/180 mg/kg bw/day groups as well as in not mated animal at 50 mg/kg bw/day.
In the female animals at 200/180 mg/kg bw/day and control groups and in the not mated female animal at 50 mg/kg bw/day of parental generation, the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
In dead and moribund animals at 100 and 200/180 mg/kg bw/day dose groups, test item induced a collapse of fluid balance due to the effect of the high doses of test item, accompanied with circulatory disturbance, incomplete O2 supply and different histological lesions in the investigated organs. These symptoms and lesions were not observed in the surviving animals in the mid (100 mg/kg bw/day) or high (200/180 mg/kg bw/day) dose groups (23/24 and 14/24, respectively).

Dead and moribund animals
Histological examinations of dead/moribund female animals revealed dilatation of stomach, small and large intestines (1/1 at 100 mg/kg bw/day, 10/10 at 200/180 mg/kg bw/day) in connection with large amount of fluid accumulation in the lumen and exfoliation of mucous membrane without significant histopathological changes (inflammation, ulceration or other lesions) as cause of death. Erosions of the mucous membrane in stomach was also observed in dead/moribund female animals (10/10 at 200/180 mg/kg bw/day)
In addition, vacuolation of hepatocytes, hepatocellular necrosis, vacuolation in the renal tubules, vacuolation in the cortical cells in adrenal glands, lymphocyte depletion in the spleen and in the thymus and alveolar emphysema in the lungs were detected in dead/moribund animals as follows:
- vacuolation of hepatocytes: 4/10 at 200/180 mg/kg bw/day;
- hepatocellular necrosis: 2/10 at 200/180 mg/kg bw/day;
- vacuolation in the renal tubules: 6/10 at 200/180 mg/kg bw/day;
- vacuolation in the cortical cells in adrenal glands: 10/10 at 200/180 mg/kg bw/day;
- lymphocyte depletion in the spleen: 1/1 at 100 mg/kg bw/day and 10/10 at 200/180 mg/kg bw/day;
- lymphocyte depletion in the thymus: 1/1 at 100 mg/kg bw/day and 10/10 at 200/180 mg/kg bw/day;
- emphysema in the lungs: 1/1 at 100 mg/kg bw/day and 10/10 at 200/180 mg/kg bw/day;
The fatal death/ moribund state of the listed animals could be in connection with the collapse of fluid balance – due to the effect of the used dose of test item – accompanied with circulatory disturbance in these animals. The vacuolation of hepatocytes, the hepatocellular necrosis and the vacuolation in the renal tubules were observed only in dead animals but not in the rats, treated with high dose of test item and euthanized at terminal sacrifice. So, these latter lesions could be rather in connection with the circulatory disturbance, the incomplete O2 supply, than the direct effects of test item. The vacuolation in the cortical cells in adrenal glands, the lymphocyte depletion in the spleen, and the alveolar emphysema in the lungs also could develop due to acute stress because of agony.
However, a test item effect cannot be excluded in the development of lymphocyte depletion in the thymus (indicative of an accelerated involution) in all dead/ moribund animals.

Surviving animals
In control and test item treated animals, erosion (5/24 control; 3/10 at 50 mg/kg bw/day, female animals) and hemorrhages (1/1 male at 50 mg/kg bw/day, 6/10 female at 50 mg/kg bw/day) in the mucous membrane of stomach in connection with the macroscopically visible focal hemorrhage were observed. These lesions could be due to mechanical origin, . i.e., local effect of the test item.
One or both sided renal pyelectasia was detected in several case: 2/24 male and 2/24 female control; 5/5 male at 50 mg/kg bw/day; 4/4 male at 100 mg/kg bw/day; 4/24 male and 2/14 female at 200/180 mg/kg bw/day) without significant histological lesions (inflammation, degeneration, fibrosis etc.) and pathological significance.
Lipoma in the abdominal cavity (1/1 control, and 1/1 at 50 mg/kg bw/day male), focal chronic inflammation in the splenic capsule (1/24 male at 200/180 mg/kg bw/day), focal fibrosis in the Glisson’s capsule (1/1 male at 50 mg/kg bw/day), cystadenoma in the pituitary (1/24 control male), and hyperplasia of BALT (Bronchus Associated Lymphoid Tissue) in the lung (1/24 male at 200/180 mg/kg bw/day) can be considered as individual disorders without toxicological significance.
Atrophy of hair follicles (1/1 male at 100 mg/kg bw/day; 1/1 female at 50 mg/kg bw/day, 1/14 female at 200/180 mg/kg bw/day) accompanied with focal or multifocal alopecia is a common finding in laboratory rats. Under conventional conditions dermal infections (Trichophyton spp, Microsporon spp, Demodex infection etc.) may occur. In these cases, dermatitis is seen. In non-infectious cases of alopecia imbalance of hormonal factors with age, malnutrition or focal ischemia could play role in development of atrophy of hair follicles and alopecia. Corticosteroids (stress) reduce the metabolic activity of epidermal cells.
The dilatation of uterus occurred in some control and treated animals: 4/24, 8/8, 4/5 and 7/14 respectively to groups of control, 50, 100 and 200/180 mg/kg bw/day. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and could be in connection with the normal sexual cycle (proestrus phase) of uterus.
There was no morphological evidence of test item related acute or subacute injuries (degeneration, inflammation, necrosis etc.) in the small and large intestines, liver, pancreas, cardiovascular system, respiratory system, immune system, hematopoietic system, skeleton, muscular system, central, or peripheral nervous system, eyes or integumentary system.
The cytomorphology of endocrine glands were the same in the control and treated animals.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Serum Levels of Thyroid Hormones
The thyroid hormone (FT3, FT4 and TSH) levels were not adversely influenced in the parental male or female animals at any dose levels.
The FT3, FT4 and TSH levels were comparable in male animals in the control, 50, 100 and 200/180 mg/kg bw/day groups.
In the female animals, the TSH level exceeded the control at 50 mg/kg bw/day.
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
The estrous cycle was irregular in several parental female animals in each group (control, 50, 100 and 200/180 mg/kg bw/day) – especially in the high dose group (50 % of the animals) – during the two last weeks of an overall of 10 weeks pre-mating period.
The examined parameters of the estrous cycle were comparable in the control and 50 and 100 mg/kg bw/day groups.
Statistical significance was noted for the lower mean number of cycles, for the lower mean number of days in pre-estrous and estrous and higher mean number of days in diestrus at 200/180 mg/kg bw/day.
A perturbation of the estrous cycle is typical in case of significant body weight reduction (ECETOC Technical report No. 31; 2002). The reproductive performance (copulatory and fertility ability) of high dose females has not been influenced by this alteration. The effects were therfore regarded as non-adverse.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Sperm examinations did not reveal any adverse test item related influence on the sperm cells at 200/180 mg/kg bw/day.
Statistical significances were detected at the lower mean percentage of immotile sperm cells in parental male animals at 200/180 mg/kg bw/day.
The mean percentage of sperms with not normal morphology (separated head and tail) were higher in the control group than in the 200/180 mg/kg bw/day.
These statistically significant differences between the control and test item treated animals were indicative of biological variation and normal function of male genital organs at 200/180 mg/kg bw/day.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The reproduction performance of male and female animals (mating and fertility) was not selectively affected by the treatment with 50, 100 or 200/180 mg/kg bw/day with respect to their control. The lower incidence of dams delivered at 100 and 200/180 mg/kg bw/day was related to the premature death around the day of birth due to severe maternal toxicity of the test item.
The copulatory index was higher than in the control group in male and female animals at 100 and 200/180 mg/kg bw/day as one control pair failed to mate.
The fertility indices were comparable in each group (male and female).
The percentage of delivered dams was significantly lower than in the control group due to the high mortality of dams at 200/180 mg/kg bw/day.
A test item effect on delivery data considered as secondary in nature was detected in dams at 200/180 mg/kg bw/day. Lowered mean number of births (total, live and viable pups) were due to maternal toxicity and high mortality of high dose treated dams close to delivery. The nursing instinct of dams was reduced at 100 and 200/180 mg/kg bw/day.
Delivery data of dams was comparable in the control, 50 and 100 mg/kg bw/day groups.
At 200/180 mg/kg bw/day, statistical significance with respect to the control was observed at the lower mean number of viable pups per litter on post-partum day 0. The mean number of total births per litter and live borns per litter was also lower than in the control group and below the historical control values (no statistical significance). Necropsy observations of dead dams at 200/180 mg/kg bw/day revealed well developed pups in the uterine horns and the mean number of pups per litter (12.7 pups/litter of dead dams) met well the actual control and historical control values.
The mean number of implantation sites, the mean number of post-implantation loss, duration of pregnancy and live birth indices were similar in the control and 200/180) mg/kg bw/day group.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Remarks on result:
other: Only treatment related effects secondary to systemic effects. Lower incidence of dams delivered at 100 and 200/180 mg/kg bw/day was related to the premature death around the day of birth.
Key result
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No effects observed regarding male reproductive performance up to the highest dose tested.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
Key result
Critical effects observed:
no
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Offspring: There were no adverse clinical signs in the F1 offspring from post-natal day 0 to 21. The percentage of offspring showing signs (no milk in the stomach, cold, found dead, missing) was higher at 200/180 mg/kg bw/day compared to the control. Most of these observations were detected on the day of delivery and corresponded with the findings of inadequate nursing behavior of the respective dams. The percentage of offspring, which were cold or did not suckle were similar in the control and 50 or 100 mg/kg bw/day groups. Some other sporadic clinical signs were also observed in the control and 50, 100 or 200/180 mg/kg bw/day dose groups (pale, cyanotic skin, smaller than others, hemorrhage on the skin).

Cohort 1A: There were no test item related clinical signs in male or female animals in F1 Cohort 1A generation in control, 50, 100 or 180 mg/kg bw/day groups.
The behavior and physical condition of all male animals were normal during the entire observation period (PND 22-PND104, 105, 106, 107, 108).
Small wounds all over the body were detected in two female animals at 50 mg/kg bw/day (2/20) as species specific sign between PND 92-109 and PND106-108. The behavior and physical condition of all other female animals were judged to be normal from birth to the termination of the study.
The behavior and physical condition of F1 Cohort 1A animals was not adversely affected by the test item at any dose level (50, 100 or 180 mg/kg bw/day) based on the weekly detailed clinical observations during the entire treatment period.
Small wounds all over the body were detected in one female animal at 50 mg/kg bw/day (1/20) on PND 98 and 105 at the weekly observations.

Cohort 1B: There were no test item related clinical signs in male or female animals in F1 Cohort 1B generation in control, 50, 100 or 180 mg/kg bw/day groups.
The behavior and physical condition of all male animals were normal during the entire observation period (PND 22-110-115).
Small wounds all over the body were detected in one female animal at 50 mg/kg bw/day (1/20) as species specific sign between PND 112-114. The behavior and physical condition of all other female animals were judged to be normal from birth to the termination of the study.
The behavior and physical condition of F1 Cohort 1B animals was not adversely affected by the test item at any dose level (50, 100 or 180 mg/kg bw/day) based on the weekly detailed clinical observations during the entire treatment period.
Small wounds all over the body were detected in one female animal at 50 mg/kg bw/day (1/20) on PND112 at the weekly observations.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Offspring: The extra uterine mortality of F1 offspring exceeded the control at 200/180 mg/kg bw/day on post-natal day 0 and between post-natal days 0 and 21. The mean number of viable pups per litter were slightly lower than in the control group at 200/180 mg/kg bw/day on post-natal day 0. However, mean value remained within the historical control. The mean number of dead pups per litter was higher than in the control at 50 and 100 mg/kg bw/day groups with statistical significance in the low dose group only from birth up to post-natal day 21. However, values of these groups remained well within the historical control ranges while the mean mortality per litter was below the historical control mean in the actual control group. Statistical significance with respect to the control was detected at the higher mean number of dead pups/ litter at 200/180 mg/kg bw/day on post-natal day 0 and from birth to post-natal day 21. Severe maternal toxicity together with bad nursing behavior may be considered when evaluating higher mortality in high dose group. The survival index was lowered compared to the control in all test item administered groups on post-natal days 4, 7 and 14.

Cohort 1A: There was no mortality in F1 Cohort 1A animals in control, 50, 100 or 180 mg/kg bw/day groups (male or female) during the course of study.

Cohort 1B: There was no test item related mortality in F1 Cohort 1B animals in control, 50, 100 or 180 mg/kg bw/day groups (male or female) during the course of study.
One female animal at 100 mg/kg bw/day (1/20) was found dead on post-natal day 57. Piloerection and decreased activity were observed before the death. Considering the low incidence of these findings and necropsy observations, the cause of the death was an individual lesion.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Offspring: The body weight development of the F1 offspring was reduced at 100 and 200/180 mg/kg bw/day.
The mean litter weight and litter weight gain were comparable with control at 50 mg/kg bw/day during the 21-day observation period.
The mean litter weight remained below the control at 100 and 200/180 mg/kg bw/day with statistical significance on post-natal days 0, 4, 7, 14 and 21. The mean litter weight gain at 100 mg/kg bw/day was significantly lower than in the control group at 100 mg/kg bw/day between post-natal days 7-14 and when summarized over days 0-21. Statistical significance was also observed at 200/180 mg/kg bw/day between post-natal days 0-4, 7-14, 14-24 and when summarized over days 0-21.
The mean body weight and body weight gain of offspring were comparable with control and at 50 mg/kg bw/day during the 21-day observation period.
Statistical significance with respect to the control was detected at the lower mean body weight of pups at 100 mg/kg bw/day on post-natal day 0 and at the lower mean body weight gain between post-natal days 7-14 and if summarized (between post-natal days 0 and 21).
The mean body weight and body weight gain of pups were below the control value at 200/180 mg/kg bw/day from birth up to post-natal day 21.
The body weight of male pups and female pups at 50 mg/kg bw/day – evaluating separately the two genders – was similar to the control on post-natal day 4.
Statistical significance with respect to the control was detected at the slightly lower mean body weight of male or female pups at 100 and 200/180 mg/kg bw/day on post-natal day 4.

Cohort 1A: The body weight development was reduced in F1 Cohort 1A animals administered with 100 and 180 mg/kg bw/day (male and female). The reduction compared to control was less than 10 % in the female animals at 100 mg/kg bw/day and was therefore considered to have little or no toxicological relevance. The mean body weight was comparable to their control in F1 Cohort 1A male animals at 50 mg/kg bw/day during the entire observation period although mean body weight gain was transiently significantly lowered compared to their control between post-natal days 91-98.
At 100 mg/kg bw/day, the mean body weight of male animals remained below the control from PND22 up to termination reaching statistical significance from PND 56 up to and including PND 105 (minus 6-10 %). The body weight gain of male animals in mid dose group was also lower than in the control in most of the weeks with statistical significance between PND42-63 by weekly interval, between PND77-84, 91-98 and if summarized between PND22-105.
Statistically significant difference with respect to the control was detected at the lower mean body weight of male animals at 180 mg/kg bw/day from weaning up to termination of the study (minus 9-18%). The mean body weight gain of these animals was also lower than in the control during the entire observation period reaching statistical significances in several cases by weekly interval (between PND22-29, 36-39, 42-70, 77-98) and also for the summarized body weight gain (between PND22-105).
The mean body weight and body weight gain was comparable in the control and test item treated F1 Cohort 1A female animals at 50 mg/kg bw/day during observation period (PND22-105).
In female animals at 100 mg/kg bw/day, lower mean body weight was observed from weaning up to PND105 with statistical significance compared to the control between PND22-39, on PND77, 91, 105 (-7 % at the lowest). The mean body weight gain of female animals in mid dose group was also slightly depressed with statistical significance with respect to the control between PND22-25 and 39-42.
In female animals at 180 mg/kg bw/day, the mean body weight was significantly lower than in the control during the entire observation period (from PND22 up to and including PND105; minus 7-11 %). The body weight gain of these female animals was also lower than in the control in most cases reaching statistical significances by weekly interval (PND22-25, 42-49, 56-63) and also for the summarized body weight gain (between PND22 and PND105).

Cohort 1B: The body weight development was reduced in F1 Cohort 1B animals administered with 180 mg/kg bw/day (male and female).
The body weight development was also depressed in male animals at 100 mg/kg bw/day from PND98 onwards.
The mean body weight and body weight gain was comparable to their control in F1 Cohort 1B male animals at 50 mg/kg bw/day during the entire observation period.
At 100 mg/kg bw/day, the mean body weight of male animals remained below the control (minus 6-7 %) consistently from PND22 up to termination, reaching statistical significance from PND 98 up to and including PND112. The mean body weight gain of these animals in mid dose group was also lower than in the control with statistical significance between PND22-25, 77-84 and 91-98 and if summarized between PND22-112.
Statistically significant difference with respect to the control was detected at the lower mean body weight of male animals at 180 mg/kg bw/day from PND25 up to termination of the study (minus 7-21 %). The mean body weight gain of these animals was also lower than in the control during the entire observation period reaching statistical significances in several cases (between PND22-25, 29-32, 36-39, 42-98 by weekly interval) and also for the summarized mean body weight gain (between PND22-105).
The mean body weight and body weight gain were comparable in the control and test item treated F1 Cohort 1B female animals at 50 mg/kg bw/day during observation period (PND22-112).
In female animals at 100 mg/kg bw/day, lower mean body weight was observed on PND25. The mean body weight gain of these female animals was comparable to their control between PND22-112.
In female animals at 180 mg/kg bw/day, the mean body weight was lower than in the control reaching statistical significances in several cases during the entire observation period (from PND22 up to and including PND112; minus 5-12 %) reaching statistical significance between PND22-36, on PND 56 and between PND77-112. The body weight gain of these female animals was also lower than in the control in most cases and reaching statistical significances between PND22-25 and also at the summarized body weight gain (between PND22 and PND112).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Offspring: not applicable

Cohort 1A: The mean daily food consumption of F1 Cohort A male and female animals was reduced at 100 and 180 mg/kg bw/day during the entir observation period in accordance with the body weight changes.
At 50 mg/kg bw/day, the mean daily food consumption of F1 Cohort 1A male animals was similar to their control during the complete observation period (between PND22 and PND105).
Statistically significant difference with respect to the control was detected at the lower mean daily food consumption in male animals at 100 mg/kg bw/day between post-natal days 49-56 and from PND77 up to the termination of the study (minus 9-14 %).
At 180 mg/kg bw/day, the mean daily food consumption of male animals was lower than in the control group from weaning up to termination (minus 4-154 %) significantly and showing statistically significant difference to the control by weekly interval between PND22-29, 36-70, 77-105.
At 50 mg/kg bw/day, the mean daily food consumption of F1 Cohort 1A female animals was comparable to their control during the observation period (between PND22 and PND105).
In the female animals at 100 mg/kg bw/day, the mean daily food consumption was reduced from weaning up to the termination of the study (6-19 %).
In the female animals at 180 mg/kg bw/day, the mean daily food consumption of female animals was lower than in the control from weaning up to the termination of the study reaching statistical significance between PND22-77 (5-19 %).

Cohort 1B: The mean daily food consumption of F1 Cohort B male and female animals was reduced at 180 mg/kg bw/day during the observation period in accordance with the body weight changes.
At 50 mg/kg bw/day, the mean daily food consumption of F1 Cohort 1B male animals was similar to their control during the observation period (between PND22 and PND112).
Statistically significant difference with respect to the control was detected transiently at the lower mean daily food consumption in male animals at 100 mg/kg bw/day between post-natal days 22-29 (-9 %).
At 180 mg/kg bw/day, the mean daily food consumption of male animals was lower than in the control group from weaning up to termination (minus 9-19 %) and reaching statistically significant difference to the control by weekly interval between PND22-42, PND49-98 and PND105-112.
In the female animals at 50 mg/kg bw/day, the mean daily food consumption was comparable with the control during the course of the entire study.
At 100 mg/kg bw/day, statistical significance was detected at the lower mean daily food consumption between post-natal day 22-29 and 42-49
(-8 %).
At 180 mg/kg bw/day, the mean daily food consumption of female animals was significantly lower than in the control group between PND29-36, 42-63 by weekly interval and between PND70-77 (minus 8-12 %).
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Offspring: not applicable

Cohort 1A: Rückmeldung von TC: There were no test item related adverse changes in the examined hematological parameters in F1 Cohort 1A male or female animals at 50, 100 or 180 mg/kg bw/day.
In the male animals at 50 mg/kg bw/day, statistical significances were detected at the slightly higher mean percentage of neutrophil granulocytes (NEU), higher mean platelet count (PLT) and at the slightly shorter mean prothrombin time (PT) when compared to the control.
In the male animals at 100 mg/kg bw/day, the mean percentage of eosinophil granulocytes (EOS) and the mean platelet count were significantly higher while the mean prothrombin time (PT) was slightly shorter than in the control group.
At 180 mg/kg bw/day, statistical significances with respect to the control were detected at the higher mean percentage of neutrophil granulocytes, monocytes (MONO), at lower mean percentage of lymphocytes (LYM) and reticulocytes (RET), lower mean corpuscular volume (MCV) elevated platelet count and shorter mean prothrombin time in male animals.
All examined hematological and blood coagulation parameters were comparable with the control in female animals at 50 mg/kg bw/day.
Statistical significance was detected with respect to the control at the slightly higher mean white blood cell count (WBC) at 100 mg/kg bw/day and at the lower mean corpuscular volume and lower mean corpuscular hemoglobin content (MCH) in female animals at 100 and 180 mg/kg bw/day. The changes noted in these hematology or blood coagulation parameters were considered to have little or no toxicological relevance. Changes in NEU, MONO along with changes in LYM in male animals may reflect a slight inflammatory process as well as stress due to malnutrition and body weight loss.
The values of WBC, MCV, MCH, PLT, RET, PT, EOS, RET and PT were well within the normal range in male or female animals, where relevant. Some of these changes – WBC, MCV, MCH, PLT, PT, EOS – were not related to doses.
There were no related changes in other hematological parameters therefore, the differences in these parameters were considered to have little or no toxicological relevance.

Cohort 1B: not applicable
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Offspring: not applicable

Cohort 1A: Pathologic alterations were not detected at the evaluation of clinical chemistry parameters in F1 Cohort 1A male or female animals at 50, 100 or 180 mg/kg bw/day.
Significantly elevated mean concentration of creatinine (CREA) in male animals at 100 and 180 mg/kg bw/day may refer to enhanced or impaired renal function. Clinical pathology, necropsy or histopathological investigations did not reveal any related changes in the examined parameters or organs. Therefore, these minor changes were judged to be of little or no toxicological significance.
In the male animals at 50 mg/kg bw/day, statistical significance with respect to the control was noted for the slightly higher mean concentration of cholesterol (CHOL), albumin (ALB) and total protein (TPROT).
At 100 mg/kg bw/day, a significantly higher mean activity of alanine aminotransferase (ALT) and elevated level of creatinine (CREA) were observed when compared to their control in male animals.
In the male animals at 180 mg/kg bw/day, statistical significance with respect to the control was noted for the higher mean activity of alanine aminotransferase, elevated levels of creatinine and cholesterol as well as at the lower mean concentrations of urea and glucose (GLUC).
The examined clinical chemistry parameters were comparable with the control in female animals of the 50 mg/kg bw/day group.
Significantly higher mean concentration of urea and lower mean concentration of albumin were detected in female animals at 100 mg/kg bw/day when compared to their control.
In the female animals at 180 mg/kg bw/day, the albumin and total protein concentrations were significantly below the control value.
The statistically significant changes of some clinical chemistry parameters were considered to be of little or no biological significance. There were no supporting histological findings in the relevant organs. Changes in enzyme activity ALT might be indicative of functional alteration in accordance with altered demands.


Cohort 1B: not applicable
Urinalysis findings:
no effects observed
Description (incidence and severity):
Offspring: not applicable

Cohort 1A: There were no test item related changes in the examined urine parameters in F1 Cohort 1A animals (male or female) at 50, 100 or 180 mg/kg bw/day. The examined urine parameters were comparable in the control and 50, 100 and 180 mg/kg bw/day groups (male and female).


Cohort 1B: not applicable
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
Offspring: The F1 offspring’s development (surface righting reflex, pinna detachment, eye opening) was depressed at 100 and 200/180 mg/kg bw/day. This finding might be related to the insufficient maternal nursing instinct mainly on PND0. F1 offspring development was clearly impaired at 100 and 200/180 mg/kg bw/day in full accordance with clinical signs and body weight changes.
There were no toxicologically relevant differences in the offspring’s development between the control and 50 mg/kg bw/day groups.
Significantly lower percentage of pups with positive response or higher percentage of pups with negative response was observed at pinna detachment, eye opening – male and female – at 100 and 200/180 mg/kg bw/day and at surface righting – female – at 200/180 mg/kg bw/day.

Cohort 1A: The sexual maturity was not adversely affected in F1 Cohort 1A male or female animals at 50, 100 or 180 mg/kg bw/day.
The balano-preputional separation was completed in all F1 Cohort 1A male animals – control, 50, 100 and 180 mg/kg bw/day – on post-natal day 25, although, the mean body weight was slightly but significantly lower with respect to the control in male animals at 180 mg/kg bw/day on PND25.
In the F1 Cohort 1A female animals, post-natal days of vaginal patency, of appearance of the first cornified vaginal smear and the interval between days of vaginal patency and first cornified smear were similar in all groups (control, 50, 100 or 180 mg/kg bw/day).

Cohort 1B: The sexual maturity was not adversely affected in F1 Cohort 1B male or female animals at 50,. 100 or 180 mg/kg bw/day.
The balano-preputional separation was completed in all F1 Cohort 1B male animals – control, 50,. 100 and 180 mg/kg bw/day – on post-natal day 25, although, the mean body weight was slightly but significantly lower with respect to the control in male animals at 180 mg/kg bw/day on PND25.
In the F1 Cohort 1B female animals, statistical significance with respect to the control was observed at the lower mean of post-natal days of vaginal patency at 100 mg/kg bw/day and at the higher mean number of post-natal days of vaginal patency at 180 mg/kg bw/day. The mean body weight was lower with respect to the control in female animals at 100 and 180 mg/kg bw/day on PND25.
The differences to the control were minor and were judged to be toxicologically not relevant as individual values corresponded to the actual control ranges.
Anogenital distance (AGD):
effects observed, treatment-related
Description (incidence and severity):
Offspring: The normalized anogenital distances were not adversely affected by the test item in male or female offspring at 50, 100 or 200/180 mg/kg bw/day. Statistical significance was detected at the shorter absolute anogenital distance of male and female pups at 100 mg/kg bw/day and in female pups at 200/180 mg/kg bw/day, however the difference disappeared when referring to the body weight.

Cohort 1A/B= not applicable
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Offspring: Nipples/areoles were not visible in any of the examined male offspring in the control or 50, 100 or 200/180 mg/kg bw/day groups on post-natal day 13.

Cohort 1A/B= not applicable
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Offspring: There were no test item related changes in the weights of examined organs (absolute and relative to body and brain weights) in male and female F1 offspring at the necropsy at weaning.
Statistical significance with respect to the control were detected at the lower mean thymus weights relative to body weight at 100 mg/kg bw/day in male offspring. Organ weights of the high dose (200/180 mg/kg bw/day) offspring are missing because not enough pups were available due to high mortality rate of dams during gestation.

Cohort 1A: The weights of the examined organs were not adversely affected in F1 Cohort 1A male and female animals at 50, 100 or 180 mg/kg bw/day.
Changes in the weight of several organs at 100 and 180 mg/kg bw/day were considered to be related to significantly reduced mean fasted body weight in male and female animals. The absolute weights of these organs were lowered and the organ weights relative to body weight were elevated when compared to the control.
At 50 mg/kg bw/day, statistically significant difference with respect to the control was observed at the lower mean brain weight and at the higher mean weight of pituitary (absolute and relative to body and brain weights), at the higher mean liver weight relative to body weight and higher mean prostate weight relative to brain weight in male animals.
In the male animals at 100 mg/kg bw/day, significantly lowered mean fasted body weight, lowered mean weights of brain, kidneys, heart, thymus, spleen and elevated mean weights relative to body weight of brain, liver, pituitary, testes and epididymides were detected when compared to the control. The mean thymus weights relative to body and brain weights were lower than in the control group while mean testes weight relative to brain weight exceeded the control.
In male animals at 180 mg/kg bw/day, significantly lowered mean fasted body weight, lowered mean weights of brain, kidneys, heart, thymus, spleen and seminal vesicles were detected when compared to their control. The mean weights relative to body weight of brain, liver, kidneys, heart, pituitary, testes, epididymides, adrenal glands, thyroids and submandibular lymph nodes were higher than in the control group. Significantly lowered mean body weight, lowered mean thymus weight and elevated mean weight of testes, each relative to brain weight, were also detected in male animals administered with 180 mg/kg bw/day.
In the female animals at 50 mg/kg bw/day group, the weight of examined organs were comparable with their control.
At 100 mg/kg bw/day, statistically significant difference with respect to the control was observed at the lower mean fasted body weight, weights of brain, kidneys, heart, thymus in female animals. The mean thymus weight relative to body and brain weights were lower and the spleen weight relative to body weight was higher than in the control group in mid dose treated female animals.
At 180 mg/kg bw/day, lower mean fasted body weight, lower mean weights of brain, kidneys, heart, thymus, pituitary and popliteal lymph node in female animals. The mean weights of brain, liver, spleen and ovaries each relative to body weight were above the control and the mean fasted body weight relative to brain weight were lower than in the control group in female animals at 180 mg/kg bw/day. The mean thymus weight relative to body and brain weights were lowered when compared to the control in these female animals.
Morphological changes were not detected during the histopathological examination. Hematology investigations as well as clinical chemistry parameters did not reveal test item related abnormalities. Therefore, changes in organ weights were judged to have little or no toxicological relevance.

Cohort 1B: The weights of the examined organs were not adversely affected in F1 Cohort 1B male and female animals at 50, 100 or 180 mg/kg bw/day.
Changes in the weight of several organs at 100 and 180 mg/kg bw/day were considered to be related to significantly reduced mean fasted body weight in male animals. The absolute weights of these organs were lowered and the organ weights relative to body weight were elevated when compared to the control.
At 50 mg/kg bw/day, statistically significant difference with respect to the control was observed at the higher mean weights of seminal vesicles (absolute and relative to body and brain weights) and at the higher mean weight of testes relative to body weight.
In male animals at 100 mg/kg bw/day, significantly lowered mean fasted body weight and lowered mean weights of brain (absolute) and higher mean weights of testes and seminal vesicles (relative to body and brain weights, both later organs) were detected when compared to the control.
At 180 mg/kg bw/day, the mean fasted body weight and mean absolute weight of most of examined organs (brain, epididymides, seminal vesicles, prostate and pituitary) were below the control value. Statistical significances with respect to the control were also noted for the higher mean weight of brain, testes and epididymides relative to body weight as well as for the lower mean fasted body weight, weights of seminal vesicles and prostate each relative to brain weight. The mean weight of testes relative to brain weight was also elevated in male animals administered with 180 mg/kg bw/day when compared to the control.
In the female animals at 50 mg/kg bw/day group, the weight of examined organs were comparable with their control.
Statistically significant difference with respect to the control was observed at the lower mean pituitary weight at 100 mg/kg bw/day as well as at the lower mean fasted body weight, lower mean weight of brain, uterus, and pituitary at 180 mg/kg bw/day in female animals of F1 Cohort 1B.
Although the above-mentioned differences with respect to the control achieved statistical significances in the weights of examined organs, these were considered to be of little or no toxicologically significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Offspring: Specific macroscopic alterations were not found in F1 offspring subjected to gross pathological examination before weaning or at weaning.
Some common sporadic necropsy findings were detected in pups subjected to necropsy before weaning:
- pale: 1/31 control; 1/44 at 100 mg/kg bw/day;
- smaller than normal: 2/31 control;
- no milk in the stomach: 11/44 at 100 mg/kg bw/day, 2/33 at 200/180 mg/kg bw/day
- autolysis of visceral organs: 4/44 at 100 mg/kg bw/day, 3/33 at 200/180 mg/kg bw/day
- missing visceral organs 6/44 at 100 mg/kg bw/day, 9/33 at 200/180 mg/kg bw/day;
- gas filled abdominal cavity: 1/33 at 100 mg/kg bw/day;
At weaning, thymic hemorrhage was detected in one male pup in the control group and right side pyelectasia was noted for one male pup at 50 mg/kg bw/day.

Cohort 1A: Macroscopic alterations related to the effect of the test item were not detected in F1 Cohort 1A male or female animals at 50, 100 or 180 mg/kg bw/day at the necropsy.
Right or both sided renal pyelectasia were observed in the control and test item administered male animals as follows: 6/20 control, 5/20 in 50 mg/kg bw/day, 3/20 in 100 mg/kg bw/day and 4/20 in 180 mg/kg bw/day groups.
Brown (1/20) and brownish-red discoloration (1/20) of the thymus was observed in male animals at 100 and 180 mg/kg bw/day, respectively.
In the female animals, necropsy observations revealed the following findings:
- control group: right or both sided pyelectasia (4/20), slight, moderate or marked hydrometra (10/20);
- 50 mg/kg bw/day: right or both sided pyelectasia (6/20), slight, moderate or marked hydrometra (10/20), wounds on the skin all over the body (2/20), brownish red color of thymus (1/20);
- 100 mg/kg bw/day: slight, moderate or marked hydrometra (7/20), missing vaginal orifice (1/20) and yellowish mucous liquid filled uterine horns (1/20);
- 180 mg/kg bw/day: right side pyelectasia (5/20), moderate or marked hydrometra (6/20), brown/ brownish red color of thymus (2/20); brownish-red discoloration of mesenteric lymph nodes (1/20);
Pyelectasia, hydrometra, brownish-red colored mesenteric lymph nodes and wounds on the skin are common findings in experimental rats of this strain and age. Hydrometra (i.e., dilatation of uterine horns with liquid content) is related to the female sexual cycle and is a physiological phenomenon. In the lack of related inflammatory or other pathological signs, these findings were judged to be toxicologically not relevant and not test item related as no dose response was noted. Discolored thymus is also a frequently observed findings in experimental rat in connection with the exsanguination procedure.
Missing vaginal orifice along with accumulation of yellowish mucous liquid content are rarely observed findings in not treated female animals of this strain. Both findings were only observed in one female at 100 mg/kg bw/day. Thus, these are considered to be species-specific changes.

Cohort 1B: Macroscopic alterations related to the effect of the test item were not detected in F1 Cohort 1B male or female animals at 50, 100 or 180 mg/kg bw/day at the necropsy.
Dead animal
In the dead female animal (1/20 at 100 mg/kg bw/day), red colored lungs, adhesion of lungs to diaphragm, red-brown spots on the liver and softer than normal liver were detected at the necropsy. These findings refer to individual lesions causing the death of this animal.
Surviving animals
In male animals, necropsy observations revealed the following findings:
- control group: right or both sided pyelectasia in the kidneys (8/20); brownish-red liquid filled kidneys (1/20); dispositioned and grey colored seminal vesicles (1/20);
- 50 mg/kg bw/day: right or both sided pyelectasia in the kidneys (9/20), brownish-red formation in adrenal glands (1/20);
- 100 mg/kg bw/day: right or both sided pyelectasia in the kidneys (6/20), hard knot in right lobe of lungs: (1/20),
- 180 mg/kg bw/day: right or both sided pyelectasia in the kidneys (7/15); liquid filled kidneys (1/15), brownish-red colored thymus: (2/15), smaller than normal thymus (1/15); smaller than normal seminal vesicles: (1/15);
In female animals, necropsy observations revealed the following findings:
- control group: both sided pyelectasia in kidneys, (1/20), brownish-red colored thymus (1/20), slight or moderate hydrometra (9/20);
- 50 mg/kg bw/day: brownish-red color of thymus (2/20), right or both sided pyelectasia in the kidneys (8/20), slight, moderate or marked hydrometra (5/20), small wounds on the skin all over the body (1/20);
- 100 mg/kg bw/day: right or both sided pyelectasia in the kidneys (3/19); slight, moderate or marked hydrometra (8/19);
- 180 mg/kg bw/day: brownish-red colored thymus (1/9), right or both sided pyelectasia (4/9), slight hydrometra (6/9);
Pyelectasia, hydrometra and wounds on the skin are common findings in experimental rats of this strain and age. Hydrometra (i.e., dilatation of uterine horns with liquid content) related to the female sexual cycle, is a physiological phenomenon. In the lack of related inflammatory or other pathological signs, these findings were judged to be toxicologically not relevant and not test item related as no dose response was noted.
Discolored thymus is also a frequently observed findings in experimental rat in connection with the exsanguination procedure.
Dispositioned or smaller than normal seminal vesicle, hard knot on the lungs and smaller than normal thymus were considered to be individual macroscopic alterations and not related to the test item.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Offspring: not applicable

Cohort 1A: Histological examinations did not reveal pathologic alterations in the organs or tissues of F1 Cohort 1A male or female animals at 180 mg/kg bw/day.
In the F1 Cohort 1A male animals in the control and 180 mg/kg bw/day groups the investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic for the sexually mature organism in all cases. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa) representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and high dose treated animals.
The histological picture of epididymides, prostate, seminal vesicles, and coagulating glands was normal in all cases as well.
In the F1 Cohort 1A female animals of the control and 180 mg/kg bw/day groups, the ovaries, uterus, cervix and vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases.
Quantitative examinations of ovaries did not reveal test item related changes in the number of developing follicles, corpora lutea or in the number of follicular atresia at the examined level of section of F1 Cohort 1A female animals at 180 mg/kg bw/day.
The mean number of primordial and primary follicles was similar in F1 Cohort 1A female animals in the control and at 180 mg/kg bw/day.
The mean number of secondary and tertiary follicles was significantly higher and the mean number of follicular atresia was lower in F1 Cohort 1A female animals at 180 mg/kg bw/day compared to the control.
The histological structure and the cellularity of pituitary with special attention on the cytomorphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control and treated male and female animals.
In several cases, dilatation of uterine horns was observed (10/20 control; 10/10 at 50 mg/kg bw/day; 1/1 at 100 g/kg bw/day; 6/20 at 180 mg/kg bw/day). This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and is in connection with the normal sexual cycle (proestrus phase) of uterus without pathological significance.
One or both sided pyelectasia was seen in several F1 Cohort 1A male and female animals: 6/20 male and 4/20 female control; 5/5 male and 4/4 female at 50 mg/kg bw/day; 3/3 male and 8/8 female at 100 mg/kg bw/day; 4/20 male and 5/20 female at 180 mg/kg bw/day. Pyelectasia without other histopathological lesions (for example degeneration, inflammation, fibrosis etc.) is considered as an individual disorder without toxicological significance.
Congestion in the thymus (1/1 male at 100 mg/kg bw/day, 1/20 male at 180 mg/kg bw/day) and acute hemorrhage in the thymus and mesenteric lymph nodes (1/20 female at 180 mg/kg bw/day, both organs) occurred sporadically and are considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguinations.
Dermal inflammation in one female animal (1/1) at 50 mg/kg bw/day was an individual lesion without toxicological significance.
There was no morphological evidence of test item related acute or subacute injury (degeneration, inflammation, necrosis etc.) in the small and large intestines, liver, pancreas, cardiovascular system, respiratory system, immune system, hematopoietic system, skeleton, muscular system, central, or peripheral nervous system, eyes, integumentary system.
The cytomorphology of endocrine glands were the same in the control and treated animals.


Cohort 1B: not applicable
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Estrous cycle

Offspring: not applicable

Cohort F1A: The estrous cycle was not adversely affected in the F1 Cohort 1A female animals at 50, 100 or 180 mg/kg bw/day groups.
The examined parameters of the estrous cycle were comparable in the control and 50 and 100 mg/kg bw/day.
Statistical significances were observed at longer mean length of cycle, lower mean number of days in pre-estrous as well as at higher mean number of days in diestrous in female animals 180 mg/kg bw/day group when compared to control. These differences with respect to the control were with minor degree for this highly variable biological system and were therefore considered to have little or no toxicological relevance. Moreover, perturbation of the estrous cycle is a usual finding when body weight is altered (ECETOC Technical report No. 31; 2002).

Cohort 1B: The estrous cycle was not adversely affected in the F1 Cohort 1B female animals at 50, 100 or 180 mg/kg bw/day groups. The examined parameters of the estrous cycle were comparable in the control and 50 and 100 mg/kg bw/day. Statistical significances were observed at the lower mean number of days in pro-estrous in female animals of the 180 mg/kg bw/day group when compared to control. This minor difference with respect to the control was considered to have no toxicological relevance regarding the observed body weight decrease in the highest dose tested.

Serum Levels of Thyroid Hormones
Offspring: The thyroid hormone (FT3, FT4 and TSH) levels were not adversely influenced in PND22 F1 offspring at any dose levels.
The FT3, FT4 and TSH levels were comparable in male animals in the control, 50, 100 and 200/180 mg/kg bw/day groups.
Slight, statistically significant difference was detected at the lower mean FT3 level at 50 mg/kg bw/day and higher mean FT4 concentrations at 100 mg/kg bw/day in PN22 offspring. In the lack of related elevation of TSH level by doses, changes in organ weight or histopathology observations, these findings were considered to be toxicologically not relevant. Slight - statistically not significant – elevation in TSH levels seemed to be dose related, however individual values were below the actual control ranges.

Cohort 1A: The thyroid hormone (FT3, FT4 and TSH) levels were not adversely influenced in the F1 Cohort 1A male or female animals at any dose levels.
There were no statistically significant differences with respect to their control in the FT3, FT4 and TSH concentrations in male or female animals at 50, 100 or 180 mg/kg bw/day levels.
In some male animals (4/10 at 50 mg/kg bw/day and 3/10 at 100 mg/kg bw/day, 2/10 at 180 mg/kg bw/day), the TSH levels exceeded the highest value of the control group. There was no dose relevancy in the incidence of these individual findings, in the mean values at 50, 100 or 180 mg/kg bw/day, in the thyroid weights or histopathology findings, therefore these changes were considered to be incidental ones and not related to the test item.
The mean FT3, FT4 and TSH levels were comparable with the control in female animals at 50, 100 or 180 mg/kg bw/day. The TSH levels of some animals (1/10 at 50 mg/kg bw/day and 2/10 at 100 mg/kg bw/day, 3/10 at 180 mg/kg bw/day) exceeded the highest value of the control group.
There was no dose relevancy in the incidence of changes in TSH individual values, in the mean values at 50, 100 or 180 mg/kg bw/day, in the thyroid weights or histopathology findings. Therefore, these changes were considered to be incidental ones and not related to the test item (male and female).

Cohort F1B: not applicable

Sperm examinations
Offspring: not applicable

Cohort 1A: Sperm examinations did not show any test item related or adverse influence on the sperm cells at 180 mg/kg bw/day.
Statistical significances were detected at the lower mean percentage of immotile sperm cells and percentage of sperms with not normal morphology (separated head and tail) in male animals at 180 mg/kg bw/day.
These statistically significant differences between the control and test item treated animals were indicative of biological variation and normal function of male genital organs at 180 mg/kg bw/day.

Cohort F1B: not applicable

Splenic Lymphocyte Subpopulation Analysis
Offspring: not applicable

There were no adverse changes in the splenic lymphocyte subpopulation in male or female animals in F1 Cohort 1A animals in 50, 100 or 180 mg/kg bw/day groups (male or female).
Statistically significant difference with respect to the control was detected at the higher mean percentage of B cells at 100 mg/kg bw/d and at the higher mean percentage of natural killer cells (NK) and cytotoxic T cells in male animals at 180 mg/kg bw/day. These minor changes were considered to be indicative of biological variation as values met well the normal ranges (NK cells: 4.99±1.82 %; Cytotoxic T cells: 31.63±5.39%). A dose response could not be observed.

Cohort F1B: not applicable
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Results of F1 (PND0 to PND21) and Cohort 1A and B (PND22 to approx. PND105-112).
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: Cohort 1A and 1B
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: reduced development (surface righting reflex, pinna detachment, eye opening)
Remarks on result:
other: Offspring
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Parental generation


Daily clinical observations


Dead and moribund animals


In the moribund male animal in the control group (1/24), enlarged, black colored lens of right-side eye, red colored hairs around right side, decreased activity and piloerection were detected between Days 115 and Day 122 and animal was subjected to euthanasia on Day 122.


In dead female animals (1/24 in mid dose; 10/24 in high dose; i.e., 4/24 moribund and 6/24 found dead), piloerection, decreased activity, diarrhea (at 100 and 200/180 mg/kg bw/day), decreased body tone and sanguineous fore limbs were observed with the highest incidence in dead and pregnant female animals at 200/180 mg/kg bw/day. Besides black discharge around the eyes, sanguineous nasal orifice, narrow eye aperture, salivation, sanguineous vaginal orifice, dulled behavior, nuzzling up bedding material and hunched back were noted for some of these high dose treated animals before death/ euthanasia.


Signs appeared between gestation days 18 and 22 and animals were found dead / were euthanized between gestation days 19 and 22 (between Days 93 and 101).


 


Table 1 Observations of dead female animals
























































































 

Observations



Incidence of observations per group (mg/kg bw/day)



100



200/180



Salivation



0/1



3/10



Piloerection



1/1



10/10



Decreased activity



1/1



10/10



Decreased body tone



0/1



6/10



Diarrhea



1/1



5/10



Black discharge around yes



0/1



3/10



Sanguineous nasal orifice



0/1



3/10



Sanguineous fore limbs



0/1



5/10



Narrow eye aperture



0/1



3/10



Sanguineous vaginal orifice



0/1



1/10



Dulled



0/1



1/10



Nuzzling up bedding material



0/1



1/10



Hunched back



0/1



1/10



Duration of signs



G21



G18-22



Day of death/ euthanasia/



G22
Day 101



G19-22
Day 93-101



Incidence = Number of animals with findings/ number of animals examined


G = gestation day


Day = study day


 


Surviving animals


In one control male animal (1/24) wounds were detected on the skin of the right side of the neck from Day 112 up to and including Day 125.


All male animals were normal at 50 mg/kg bw/day during the entire treatment period.


Salivation (2/24) and alopecia (1/24) on the fore-limbs and chest and abdomen were observed in male animals at 100 mg/kg bw/day between Days 25 and 38 and Days 84 to Day 134, respectively.


200 mg/kg bw/day dose of the test item induced decreased activity, decreased body tone, diarrhea, piloerection or sanguineous nasal orifice between Days 4 and 10. Decreased activity and decreased body tone were also observed in two male animals (2/24) of high dose group at the end of the treatment period (from Day 116 up to and including Day 124). After the dose reduction to 180 mg/kg bw/day, salivation and nuzzling up the bedding material were seen with variable occurrence between Days 21 and 132/ 133. The lens of the right-side eye was opalescent and enlarged in one animal of the high dose group.


Table 2 summarizes the daily observations of male animals at 200/180 mg/kg bw/day.


 


Table 2 Observations of male animals at 200/180 mg/kg bw/day














































Observations



Incidence of observations at 200/180 mg/kg bw/day



Decreased activity



7/24



Decreased body tone



6/24



Salivation



15/24



Diarrhea



4/24



Piloerection



5/24



Sanguineous nasal orifice



2/24



Salivation



15/24



Nuzzling up bedding material



15/24



Opalescent and enlarged eye



1/24



Incidence = Number of animals with findings/ number of animals examined


 


In female animals, opalescent lens of the right-side eye (control group, 1/2 non-pregnant female between Days 91 and 119) and alopecia (50 mg/kg bw/day; 2/24; under the ears in one of them from Day 24 up to and including gestation day 20; on the abdomen in the other one, from gestation day 11 up to and including lactation day 24).


In the female animals at 100 mg/kg bw/day, decreased activity, piloerection, diarrhea, sanguineous nasal orifice or sanguineous fore limbs were detected during the last few days of gestation (G17-21) and first three days of lactation (L0-L2). Additionally, daily clinical observations revealed salivation with variable incidence during the pre-mating, gestation and lactation periods, as well as alopecia on the skin (at pera or chest) during the lactation period.


At 200/180 mg/kg bw/day, salivation, decreased activity, piloerection and nuzzling up the bedding material were noted for some female animals during the pre-mating period. Decreased activity, piloerection, decreased body tone, diarrhea, black discharge around eyes, sanguineous nasal orifice, and sanguineous fore limbs were detected during the last few days of gestation (G17-21) and first few days of lactation (L0-L3). Three female animals salivated after the treatment during the gestation period. Dark-red vaginal orifice, signs of bites at pera and tail, sanguineous fore limbs and alopecia were seen in single dams of the high dose group during the lactation period.


Salivation and nuzzling up the bedding material appeared immediately after the administration and ceased shortly thereafter. Therefore, these signs were considered to be not adverse.


Alopecia on the skin is a species-specific finding, which is also observed in untreated experimental rats of this strain with similar age. These were individual findings with low incidence and were thus not considered related to the treatment.


Eye alterations (opalescent, enlarged) were individual findings occurring also in non-treated animals of this strain.


Table 3 summarizes the daily observations of female animals at 100 and 200/180 mg/kg bw/day during pre-mating, gestation and lactation periods.


 


Table 3: Observations of female animals at 100 and 200/180 mg/kg bw/day during pre-mating, gestation and lactation periods








































































































































Observation



Incidence of observations



Pre-mating



Gestation



Lactation



100



200/180



100



200/180



100



200/180



mg/kg bw/day



mg/kg bw/day



mg/kg bw/day



Salivation



6/24



8/24



3/20



3/12



1/20



0/12



Decreased activity



0/24



1/24



6/20



7/12



2/20



5/12



Piloerection



0/24



2/24



9/20



7/12



4/20



5/12



Nuzzling up bedding material



0/24



2/24



0/20



1/12



0/20



0/12



Decreased body tone



0/24



0/24



0/20



2/12



0/20



3/12



Diarrhea



0/24



0/24



4/20



2/12



1/20



1/12



Black discharge around eyes



0/24



0/24



0/20



1/12



0/20



0/12



Sanguineous nasal orifice



0/24



0/24



1/20



1/12



0/20



0/12



Sanguineous fore limbs



0/24



0/24



2/20



2/12



0/20



1/12



Dark-red vaginal discharge



0/24



0/24



0/20



0/12



0/20



1/12



Signs of bites at pera and tail



0/24



0/24



0/20



0/12



0/20



1/12



Alopecia



0/24



0/24



0/20



1/12



2/20



2/12



 


Necropsy


Dead and moribund animals
In the moribund male animal in the control group (1/24), individual lesions were observed in the pituitary gland (not visible, coated by a soft yellowish-white tissue) and as consequences or related findings, enlarged dark colored separated eye lens, dilated orbit, yellow colored swollen harderian gland, smaller than normal spleen and liver, flattened brain and empty stomach were also seen at the necropsy. This kind of change in the pituitary gland occurs in non-treated experimental rats – it occurred in one control rat – and has no toxicological relevance in this study.
In the dead pregnant female animal at 100 mg/kg bw/day (1/21), dilated stomach with thin wall, accumulation of liquid or gas in the stomach or intestines, greyish-green sediment in the stomach, sign of diarrhea (thin faces around anus), dark colored mesenteric lymph nodes, smaller than normal thymus and spleen, pea sized yellowish-grey spots on the liver lobes, dark-red colored renal medulla and 13 dead fetuses were observed.
In dead and moribund pregnant female animals at 200/180 mg/kg bw/day (10/24; 4/24 moribund and 6/24 found dead), macroscopic findings were detected at necropsy as follows:
- stomach: dilated, thin wall, filled with liquid/ food, hemorrhage, greyish/greenish-brown colored, greyish-green mucous sediment, greenish colored content, thickened mucous membrane, erosions
- intestines: dilated, filled with liquid, thin wall;
- cecum: filled with yellowish-green mucous liquid, dilated, thin wall;
- thymus: smaller than normal;
- kidneys: dark brownish-red, dark colored medulla, congestive medulla
- liver: spots on the surface, dark red-black margins of lobes, smaller than normal,
- mesenteric lymph nodes: dark colored
- adrenal glands: larger than normal
- spleen: smaller than normal, black colored and destructed structure;
- hairs: sanguineous around mouth, discharge around the eyes or vaginal orifice, thin faces or translucent discharge around anus;
- fore-limbs: bleeding wounds
- Uterine horns: dead fetuses, early death, late mortality, fetus in the birth canal;
The visceral organs were slightly autolyzed in each dead pregnant animal.


The incidence of necropsy findings is summarized in Table 4.


 


Table 4 Necropsy observations of dead female animals at 200/180 mg/kg bw/day












































































































































































Organs



Observations



Incidence



Stomach



Dilated



8/10



Thin wall



9/10



Filled with liquid



6/10



Filled with liquid and food



2/10



Hemorrhage



3/10



Greyish/greenish-brown colored



3/10



Greyish-green mucous sediment



3/10



Greenish colored content



3/10



Thickened mucous membrane



1/10



Erosions



1/10



Intestines



Dilated



8/10



Filled with liquid



10/10



Thin wall



2/10



Cecum



Filled with yellowish-green mucous liquid



4/10



Dilated, thin wall



1/10



Thymus



Smaller than normal



7/10



Kidneys



Dark brownish-red



1/10



Dark colored medulla



4/10



Congestive medulla



1/10



Liver 



Spots on the surface



4/10



Smaller than normal



1/10



Dark red-black margins of lobes



1/10



Mesenteric l. n.



Dark colored



6/10



Adrenal glands



Larger than normal



4/10



Spleen 



Smaller than normal



9/10



Black colored and destructed structure



1/10



Hairs



Sanguineous around mouth



1/10



Discharge around eyes



2/10



Discharge around vaginal orifice



5/10



Thin faces around anus



4/10



Translucent discharge around anus



1/10



Fore-limbs



Bleeding wounds



4/10



Uterine horns



Dead fetuses



10/10



Early death



1/10



Late mortality



1/10



Fetus in the birth canal



2/10



Visceral organs



Autolysis



6/10




Necropsy findings of dead and moribund female animals refer to local effects of the test item in gastrointestinal tract, which led to systemic changes, acute stress and agony (contaminations on hairs around orifices, changes in, liver, kidneys, mesenteric lymph nodes, adrenal gland, spleen). Bleeding wounds on the fore limbs may refer to self-biting as a pain reaction.


 


Surviving animals


The following necropsy findings were detected in surviving male animals:


Control group: right side pyelectasia (2/23), compact fatty tissue in the abdominal cavity (1 cm x 0.5 cm; 1/23)


50 mg/kg bw/day: right or left side pyelectasia (5/24), point-like hemorrhage in the stomach (1/24), compact fatty tissue in the abdominal cavity (1 cm x 0.5 cm. 1/24); pea-sized Hernia diaphragmatica (1/24);


100 mg/kg bw/day: right or both sided pyelectasia (4/24), alopecia on the right-side fore-limbs (1/24);


200/180 mg/kg bw/day: right side pyelectasia (4/24), pale areas on the lung lobes (1/24); adhesion of the spleen to the abdominal wall (1/24);


In surviving female animals, necropsy observations revealed the following findings:


Control group: right side pyelectasia (1/21 dam, 1/2 non-pregnant female), hemorrhage in the stomach (7/21 dam), hydrometra (3/21 dam, 1/2 non-pregnant female), white lens in right side eye (1/2 non-pregnant female);


50 mg/kg bw/day: hemorrhage in the stomach (10/21 dam), hydrometra (4/21 dam), alopecia on the skin of right-side hind limb (1/21);


100 mg/kg bw/day: moderate hydrometra (2/20 dam and 2/3 non-pregnant female), additional dislocated part of spleen (1/20 dam);


200/180 mg/kg bw/day: right or both sided pyelectasia (1/12 dam 1/2 non-pregnant female), white spots on the lung lobes (1/12 dam); hemorrhages in the stomach (1/12 dam), hydrometra (5/12 dam, 2/2 non-pregnant female), alopecia on the skin of chest and abdomen (1/12 dam);


Hemorrhage in the stomach mucosa were probably due to the local effect of treatment procedure. There was no dose response relationship in the incidence of these findings or related changes. Therefore, changes in the stomach mucosa were considered toxicologically not relevant.


Hydrometra (i.e., dilatation of uterine horns), related to the female sexual cycle, alopecia and pyelectasia are common observations in experimental rats. In the lack of related inflammatory or other pathological signs, these findings were judged to be toxicologically not relevant and not test item related as no dose response was noted.


Compact fatty tissue in the fatty tissue of abdominal cavity, Hernia diphragmatica and white eye lens are individual macroscopic findings frequently observed in experimental rats of this strain with similar age. These occurred with low incidence and were considered to be toxicologically not relevant.

Conclusions:
Under the conditions of this EOGRTS, Diammonium Peroxodisulphate (APS) caused death of pregnant female animals, clinical signs, reduced body weight and food consumption (male and female), changes in the gastro-intestinal tract – supported by histological findings – in dead pregnant female animals at 200/180 mg/kg bw/day of P generation. The copulatory and fertility ability of male and female animal were not selectively influenced at 200/180 mg/kg bw/day. There was a clearly lower number of dams delivered due to premature death related to severe maternal toxicity at 200/180 mg/kg bw/day.
At 100 mg/kg bw/day of P generation, mortality of one dam, clinical signs, reduced body weight and food consumption (male and female), macroscopic and histopathological findings (only in dead animal) were observed. The copulatory and fertility ability (male or female animals) was considered normal. One pregnant female did not deliver due to premature death at 100 mg/kg bw/day.
At 50 mg/kg bw/day of P generation, there were no signs of systemic toxicity or influence on the reproductive performance (male or female animals)
In F1 offspring, reduced development (surface righting reflex, pinna detachment, eye opening) and depressed body weight development were observed at 100 and 200/180 mg/kg bw/day.
At 50 mg/kg bw/day of F1 offspring, no test item related effects were detected.
F1 adult (F1 Cohort 1A, Cohort 1B)
Reduced body weight development and food consumption were detected at 100 and 180 mg/kg bw/day in F1 adult animals (male and female). The degree of alterations at 100 mg/kg bw/day were minor and were considered to be toxicologically not relevant.
At 50 mg/kg bw/day of F1 adults, no test item related effects were detected.
Executive summary:

The subject of this study was investigating the reproductive toxicity of Diammonium Peroxodisulphate (APS) by conducting the Extended One-Generation Reproduction Toxicity Study in the rat according to OECD 443 as requested by the European Chemical Agency (ECHA). The guideline is designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing.


The aim of the one-generation reproduction toxicity study was to provide an evaluation of the pre- and post-natal effects of the test item Diammonium Peroxodisulphate (APS) on development as well as a thorough evaluation of systemic toxicity in male and female animals, in pregnant and lactating females and in young and adult offspring when repeatedly administered orally (by gavage) to animals at doses of 50, 100 and 200/180 mg/kg bw/day* compared to control animals. Adult offspring were treated at doses of 50, 100 and 180 mg/kg bw/day.


The effect of the test item was examined on the male and female reproductive performance, such as gonadal function, estrous cycle, mating behavior, conception, parturition, gestation, lactation and weaning (P generation) and on the offspring viability, neonatal health and mortality, growth and development of the offspring (F1 generation; Cohort 1A and Cohort 1B) to adulthood following oral (by gavage) administration.


Four groups of Han:WIST rats (n= 24/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 50, 100 and 200/180 mg/kg bw/day doses corresponding to concentrations of 0, 20, 60 and 200 mg/mL in parental generation. The application volume was 5 mL/kg bw. Control animals received the vehicle, sunflower oil, only.


The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). Concentration of the test item in the dosing formulations varied in the acceptable range between 93 % and 106 % of the nominal values and formulations were homogenous thereby confirming the proper dosing of parental animals (P) and F1 generations.


All animals of the parent (P) generation were dosed prior to mating (10 weeks) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 133-135 days). Dams were exposed through the mating and gestation periods and at least up to lactation day 21 (altogether for 120, 121 days in the most cases; single dams were exposed for 115, 122 or 125 days). Not mated and non-pregnant females were administered for 120 or 121 days.


Clinical observations (clinical signs, body weight, food consumption, estrous cycle) and pathology (clinical and organ pathology) examinations were performed on parental (P) animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive systems.


Estrous cycle was monitored by examining vaginal smears before the mating for two weeks and during the mating period until evidence of mating and on the day of the necropsy.


The dams were allowed to litter and rear their offspring up to day 21 post-partum.


All F1 offspring were observed individually for the health, growth, development and function up to and including post-natal day 21 (clinical signs, body weight, surface righting reflex, pinna detachment, eye opening, anogenital distance, nipple retention).


Twenty F1 animals/sex/group were randomly selected for Cohort 1A in each group and twenty F1 animals/sex/group were selected for Cohort 1B in the control, low and mid dose group on post-natal day 21 for follow-up examinations. Fifty male and eight female offspring were selected for Cohort 1B in high dose group because of the low offspring number.


Dosing of F1 offspring selected for follow-up examinations (Cohort 1A and Cohort 1B) begun on post-natal day 22 and treatment was continued up to the day before the necropsy.


F1 offspring (Cohort 1A and Cohort 1B) were observed identically to parental animals – clinical signs, body weight, food consumption, estrous cycle, clinical pathology and organ pathology. Sexual maturity of offspring was investigated by observation of balano-preputional separation, vaginal patency (Cohort 1A and Cohort 1B) and appearance of first cornified vaginal smear (Cohort 1A).


Cohort 1A animals were subjected to necropsy, organ weighing and sperm analysis – one day after the termination of the exposure – on PND 105-110.


Cohort 1B animals were observed identically to parental (P) animals and were subjected to necropsy and organ weighing on PND 111-116.


Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4 and TSH) from 10 parent (P) animals/sex/ group at termination, in surplus pups at PND 4 (pooled by litters), from F1 pups not selected for cohorts on PND 22 and from 10 adult F1 Cohort 1A male and female animals/group at termination. Thyroid hormone levels were determined in adult animals (P, F1 Cohort 1A) and in PND22 F1 offspring.


All adult animals (P, F1 Cohort 1A and Cohort 1B) were subjected to gross pathology with complete tissue preservation one day after the last treatment. Brain, spleen, thymus and mammary tissues were preserved for 10 male and 10 female pups per group – where feasible – in F1 offspring not selected for Cohorts on PND22 or shortly thereafter.


Special attention was paid to the organs and tissues of the reproductive system for P or F1 animals.


Selected organ weights were determined in adult animals (P, F1) and in offspring (PND22 or shortly thereafter).


Sperm parameters were determined in all control and high dose male animals in P generation and in F1 generation (Cohort 1A).


Full histopathology examinations were performed on the organs and tissues of adult animals (P, F1 Cohort 1A) in control and high dose groups with special emphasis on sexual organs and tissues.


Reproductive organs were also processed and examined histologically in non-mated and non-pregnant female animals and their mating partners in the low and mid dose groups.


In addition, organs showing macroscopic changes were also processed and examined histologically in adult animals in low or mid dose groups (P, F1 Cohort 1A) and in F1 offspring.


A quantitative evaluation of primordial, small growing (secondary and tertiary) follicles, as well as corpora lutea was performed in F1 Cohort 1A female animals in the control and 180 mg/kg bw/day groups.


The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only.


 


Results


Mortality


Test item related mortality was detected in pregnant female animals (P) at 100 mg/kg bw/day (1/24) and 200/180 mg/kg bw/day (10/24) close to the delivery. Based on necropsy and histopathological findings, the collapse of fluid balance caused the death/ moribund state of these pregnant female animals.


One male animal in the control group of the parental generation (1/24) died because of an individual disease.


There was no test item related mortality in F1 Cohort 1A or F1 Cohort 1B animals in control, 50, 100 or 180 mg/kg bw/day groups (male or female) during the course of study. One female animal at 100 mg/kg bw/day (1/20 Cohort 1B) was found dead on post-natal day 57 due to individual lesion.


 


Clinical observation


Female animals at 100 mg/kg bw/day and male and female animals at 200/180 mg/kg bw/day showed test item related clinical signs referring to systemic toxicity during the course of daily clinical observations.


The main clinical signs appeared at two peak-periods in male animals at 200/180 mg/kg bw/day: before the dose reduction (decreased activity, decreased body tone, diarrhea, piloerection or sanguineous nasal orifice) and at the end of the treatment period (decreased activity and decreased body tone).


In female animals at 100 and 200/180 mg/kg bw/day, the main signs (decreased activity, piloerection, decreased body tone, diarrhea, black discharge around eyes, sanguineous nasal orifice, and sanguineous fore limbs) were detected during the last few days of gestation (G17-21) and first two days of lactation (L0-L1).


Salivation at 100 and 200/180 mg/kg bw/day (male and female) and nuzzling up the bedding material at 200/180 mg/kg bw/day (male and female) were also treatment related sign but were considered to be toxicologically not relevant because of short duration after the test item administration.


Detailed weekly clinical observations also revealed some of these signs in male animals at 200/180 mg/kg bw/day on Day 7 and Day 119 and in female animals at 100 and 200/180 mg/kg bw/day on gestation day 21 and lactation day 0.


Clinical signs related to the test item effect were not detected in male or female animals in F1 Cohort 1A or Cohort 1B generation in 50, 100 or 180 mg/kg bw/day groups at the daily or weekly clinical observations.


 


Body weight and body weight gain


The body weight development was significantly reduced in parental male animals administered with 100 and 200/180 mg/kg bw/day during the entire treatment period and in female animals at 200/180 mg/kg bw/day during gestation period.


Slightly lowered mean body weight of male animals at 50 mg/kg bw/day during the entire treatment period and in female animals at 100 mg/kg bw/day during the gestation period were judged to be toxicologically not relevant because the effect could not be observed in the F1 adult generation and the difference with respect to the control remained less than 10 % both in male and female animals.


The body weight development was reduced in F1 Cohort 1A animals administered with 100 or 180 mg/kg bw/day (male or female).


Similarly, the body weight development was depressed in F1 Cohort 1B animals administered with 180 mg/kg bw/day (male and female).


 


Food consumption


The food consumption was significantly reduced in parental male (pre-mating and post-mating periods) and female animals (pre-mating, gestation and lactation periods) at 100 and 200/180 mg/kg bw/day. The mean daily food consumption of male and female animals was reduced at 100 mg/kg bw/day (Cohort 1A) and at 180 mg/kg bw/day (Cohort 1A and 1B) during the observation period in accordance with the body weight changes.


 


Estrous cycle


The estrous cycle was not adversely affected in all female animals (P, F1A, F1A) at 50, 100 or 200/180 mg/kg bw/day.


 


Delivery data of dams


Lowered mean number of births (total, live and viable pups) as a secondary test item effect on delivery data was observed in dams at 200/180 mg/kg bw/day due to maternal toxicity and high mortality of 200/180 mg/kg bw/day dose treated dams close to the delivery.


 


Reproductive performance


The copulatory performance and fertility of male and female animals (mating and fertilization) were not selectively affected by the treatment with 50, 100 or 200/180 mg/kg bw/day. The lower incidence of dams delivered at 100 and 200/180 mg/kg bw/day was related to the premature death around the day of birth due to severe maternal toxicity of the test item.


 


Urinalysis


The examined urine parameters were not adversely affected in male and female animals at 50, 100 or 200/180 mg/kg bw/day of the parental generation. There were also no test item related changes in the examined urine parameters in F1 Cohort 1A animals (male or female) at 50, 100 or 180 mg/kg bw/day.


 


Clinical pathology


There were no test item related adverse changes in the examined clinical pathology parameters in parental male or female animals at 50, 100 or 200/180 mg/kg bw/day. Elevation of mean activity of alanine aminotransferase at 100 mg/kg bw/day (male) and 200/180 mg/kg bw/day (male and female) as well as lowered albumin and total protein concentrations at 100 or 200/180 mg/kg bw/day (male or female) might be indicative of changed renal or hepatic function as no morphological changes were detected in these organs.


Pathologic alterations were not detected at the evaluation of clinical pathology (urine, hematology and blood coagulation, clinical chemistry) parameters in F1 Cohort 1A male or female animals at 50, 100 or 180 mg/kg bw/day.


 


Thyroid hormones


The serum thyroid hormone (FT3, FT4 and TSH) levels were not adversely influenced in the parental male or female animals, in PND22 F1 offspring and in the F1 Cohort 1A at any dose levels.


 


Necropsy


Necropsy observations revealed changes in the gastro-intestinal tract (stomach, intestines, cecum) in dead pregnant female animals at 100 and 200/180 mg/kg bw/day. Additionally, related signs of these changes and/or agony were detected in the liver, kidneys, spleen, adrenal glands, lungs and on the hairs of these animals.


Macroscopic alterations related to the effect of the test item were not detected in surviving parental male or female animals as well as in F1 Cohort 1A or in F1 Cohort 1B (male or female) animals in all dosing groups at the necropsy.


 


Organ weight


The weights of examined organs were not adversely affected in male or female parental animals at 50, 100 and 200/180 mg/kg bw/day. A test item influence cannot be excluded in development of the lower mean weights of thymus and prostate (male) and higher mean weights of adrenal glands (female) at 200/180 mg/kg bw/day. Cellular damage was not detected at the histopathological examination of these organs and hematology investigations as well as clinical chemistry parameters did not reveal test item related abnormalities. Therefore, weight changes of these organs were judged to be of no toxicological relevance.


The weights of the examined organs were not adversely affected in F1 Cohort 1A or F1 Cohort 1B (male and female) animals at 50, 100 or 180 mg/kg bw/day.


 


Sperm examinations


Sperm examinations did not reveal any test item related influence on the sperm cell morphology and motility, and total sperm count at 200/180 mg/kg bw/day (parental (P) and cohort 1A male animals).


 


Histopathology


Histological examinations revealed of changes in the gastrointestinal tract of dead and moribund female animals (dilatation of stomach, small and large intestines along with large amount of fluid accumulation in the lumen) and exfoliation of mucous membrane – as cause of death –without essential histopathological changes (inflammation, ulceration or other pathological lesions) at 100 and 200/180 mg/kg bw/day. These lesions were not observed in the surviving animals in the mid or high dose groups.


Thymic lymphocyte depletion – indicative of accelerated involution of the organ – in all high dose treated female animals was also considered to be related to test item.


The investigated organs of reproductive system (testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, oviduct, uterus cervix, vagina) were histologically normal and characteristic of the sexually mature organism in all parental (P) animals at 200/180 mg/kg bw/day (including not mated and non-pregnant female animals and their mating partners).


Histological examinations did not reveal pathological alterations in the organs or tissues of F1 Cohort 1A male or female animals at 180 mg/kg bw/day.


 


Splenic Lymphocyte Subpopulation Analysis


There were no test item related changes in the splenic lymphocyte subpopulation in male or female animals in F1 Cohort 1A animals at 50, 100 or 180 mg/kg bw.


 


Offspring


The extra uterine mortality of F1 offspring exceeded the control at 200/180 mg/kg bw/day on post-natal day 0 and between post-natal days 0 and 21. Severe maternal toxicity was considered when evaluating the higher mortality in high dose group.


There were no adverse clinical signs in the F1 offspring from post-natal day 0 to 21. However, the common findings of pups (no milk in the stomach, cold body temperature, percent of dead or missing pups) were observed at a higher incidence than usual at 200/180 mg/kg bw/day corresponding with the inadequate nursing behavior of the respective dams.


The F1 offspring’s development (surface righting reflex, pinna detachment, eye opening) was depressed at 100 and 200/180 mg/kg bw/day most likely due to inadequate nursing behavior of the dams.


The body weight development of the F1 offspring was slightly reduced at 100 and 200/180 mg/kg bw/day.


Specific macroscopic alterations were not found in F1 offspring subjected to gross pathological examination before weaning.


There were no test item related changes in the weights of examined organs (absolute and relative to body and brain weights) in male or female offspring at weaning.


 


In conclusion, Diammonium Peroxodisulphate (APS) caused death of pregnant female animals, clinical signs, reduced body weight and food consumption (male and female), changes in the gastro-intestinal tract – supported by histological findings – in dead pregnant female animals at 200/180 mg/kg bw/day of P generation. The copulatory and fertility ability of male and female animal were not selectively influenced at 200/180 mg/kg bw/day. There was a clearly lower number of dams delivered due to premature death related to severe maternal toxicity  at 200/180 mg/kg bw/day.


At 100 mg/kg bw/day of P generation, mortality of one dam, clinical signs, reduced body weight and food consumption (male and female), macroscopic and histopathological findings (only in dead animal) were observed. The copulatory and fertility ability (male or female animals) was considered normal. One pregnant female did not deliver due to premature death at 100 mg/kg bw/day.


At 50 mg/kg bw/day of P generation, there were no signs of systemic toxicity or influence on the reproductive performance (male or female animals)


In F1 offspring, reduced development (surface righting reflex, pinna detachment, eye opening) and depressed body weight development were observed at 100 and 200/180 mg/kg bw/day.


At 50 mg/kg bw/day of F1 offspring, no test item related effects were detected.


F1 adult (F1 Cohort 1A, Cohort 1B)


Reduced body weight development and food consumption were detected at 100 and 180 mg/kg bw/day in F1 adult animals (male and female). The degree of alterations at 100 mg/kg bw/day were minor and were considered to be toxicologically not relevant.


At 50 mg/kg bw/day of F1 adults, no test item related effects were detected.


Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:


NOAEL for systemic toxicity (male and female):  50 mg/kg bw/day


NOAEL for reproductive performance (male):     180 mg/kg bw/day


NOAEL for reproductive performance (female):   50 mg/kg bw/day


NOAEL for F1 Offspring:                                  50 mg/kg bw/day


NOAEL for F1 Adults:                                      100 mg/kg bw/day


 


 


* The high dose of 200 mg/kg bw/day was reduced to 180 mg/kg bw/day on Day 7 – after two days washing out period on Days 5 and 6 – because of clinical signs of toxicity and body weight loss of animals.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study was conducted in accordance with GLP regulation and the respective OECD/EU guideline and is considered acceptable for the assessment of effects on fertility of the test substance.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

supporting pre-studies
Several pre-studies had to be considered before performing the main study for the extended one generation reproduction toxicity (EOGRT). According to OECD guideline 443 the test protocol for EOGRT study was designed for administration of the test substance in diet which is the preferred route. The available dietary studies (with APS and SPS please refer to IUCLID sections 7.5.1 and 7.8.1) both emphasized palatability problems due to the treated diet. Thus, a 14-day palatability pre-test was performed at 250 and 500 mg/kg bw/d of APS in diet to check for possible palatability issues for the main study (IUCLID section 7.5.1; study no. 552-400-4688). Animals did not show any severe signs of toxicity and also no palatability issues. Thus, analytical method development was initiated. However the method validation showed that the substance seemed to be unstable in the diet. For diet preparation water had to be added by the feed producer. APS seem to react with the diet in presence of water. This led to a very low recovery rate. Therefore, it was judged that application via the diet is not possible and gavage was chosen as main application route for the EOGRT study.
As no data for the gavage application was available, a DRF (similar to OECD 421) for the OECD 443 was performed with 50, 100 and 250 mg/kg bw/d (IUCLID section 7.8.1; study no. 552-421-5133). At 250 mg/kg bw/d four male animals died on Days 4 and 5 (macroscopic and histopathology changes in stomach and intestines were observed during necropsy). Depressed body weight/food consumption and clinical signs were observed for all surviving male and female animals. Therefore the high dose was lowered to 180 mg/kg bw/d. At this dose the animals recovered completely. Only the body weight development of the male animals stayed slightly behind the control animals until the end of the DRF. An additional DRF study for 14 days was performed using 200 mg/kg bw/d in order to find a dose that causes toxicological effects but not severe suffering as required by the guideline (IUCLID section 7.8.1; study no. 552-421-5133R). At this dose level the animals showed no signs of toxicity within the 14 days study period. Due to the steep dose response curve (no effects at 200 mg/kg bw/d and mortality at 250 mg/kg bw/d). It was decided to use 200 mg/kg bw/d as the highest dose in the EOGRT study.
 
Main study (key, OECD 443)
The subject of this study was investigating the reproductive toxicity of (Diammonium Peroxodisulphate (APS)) by conducting the Extended One-Generation Reproduction Toxicity Study in the rat according to OECD 443 as requested by the European Chemical Agency (ECHA). The guideline is designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing.
The aim of the one-generation reproduction toxicity study was to provide an evaluation of the pre- and post-natal effects of the test item Diammonium Peroxodisulphate (APS) on development as well as a thorough evaluation of systemic toxicity in male and female animals, in pregnant and lactating females and in young and adult offspring when repeatedly administered orally (by gavage) to animals at doses of 50, 100 and 200/180 mg/kg bw/day* compared to control animals. Adult offspring were treated at doses of 50, 100 and 180 mg/kg bw/day.
The effect of the test item was examined on the male and female reproductive performance, such as gonadal function, estrous cycle, mating behavior, conception, parturition, gestation, lactation and weaning (P generation) and on the offspring viability, neonatal health and mortality, growth and development of the offspring (F1 generation; Cohort 1A and Cohort 1B) to adulthood following oral (by gavage) administration.
Four groups of Han:WIST rats (n= 24/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 50, 100 and 200/180 mg/kg bw/day doses corresponding to concentrations of 0, 20, 60 and 200 mg/mL in parental generation. The application volume was 5 mL/kg bw. Control animals received the vehicle, sunflower oil, only.
The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). Concentration of the test item in the dosing formulations varied in the acceptable range between 93 % and 106 % of the nominal values and formulations were homogenous thereby confirming the proper dosing of parental animals (P) and F1 generations.
All animals of the parent (P) generation were dosed prior to mating (10 weeks) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 133-135 days). Dams were exposed through the mating and gestation periods and at least up to lactation day 21 (altogether for 120, 121 days in the most cases; single dams were exposed for 115, 122 or 125 days). Not mated and non-pregnant females were administered for 120 or 121 days.
Clinical observations (clinical signs, body weight, food consumption, estrous cycle) and pathology (clinical and organ pathology) examinations were performed on parental (P) animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive systems.
Estrous cycle was monitored by examining vaginal smears before the mating for two weeks and during the mating period until evidence of mating and on the day of the necropsy.
The dams were allowed to litter and rear their offspring up to day 21 post-partum.
All F1 offspring were observed individually for the health, growth, development and function up to and including post-natal day 21 (clinical signs, body weight, surface righting reflex, pinna detachment, eye opening, anogenital distance, nipple retention).
Twenty F1 animals/sex/group were randomly selected for Cohort 1A in each group and twenty F1 animals/sex/group were selected for Cohort 1B in the control, low and mid dose group on post-natal day 21 for follow-up examinations. Fifty male and eight female offspring were selected for Cohort 1B in high dose group because of the low offspring number.
Dosing of F1 offspring selected for follow-up examinations (Cohort 1A and Cohort 1B) begun on post-natal day 22 and treatment was continued up to the day before the necropsy.
F1 offspring (Cohort 1A and Cohort 1B) were observed identically to parental animals – clinical signs, body weight, food consumption, estrous cycle, clinical pathology and organ pathology. Sexual maturity of offspring was investigated by observation of balano-preputional separation, vaginal patency (Cohort 1A and Cohort 1B) and appearance of first cornified vaginal smear (Cohort 1A).
Cohort 1A animals were subjected to necropsy, organ weighing and sperm analysis – one day after the termination of the exposure – on PND 105-110.
Cohort 1B animals were observed identically to parental (P) animals and were subjected to necropsy and organ weighing on PND 111-116.
Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4 and TSH) from 10 parent (P) animals/sex/ group at termination, in surplus pups at PND 4 (pooled by litters), from F1 pups not selected for cohorts on PND 22 and from 10 adult F1 Cohort 1A male and female animals/group at termination. Thyroid hormone levels were determined in adult animals (P, F1 Cohort 1A) and in PND22 F1 offspring.
All adult animals (P, F1 Cohort 1A and Cohort 1B) were subjected to gross pathology with complete tissue preservation one day after the last treatment. Brain, spleen, thymus and mammary tissues were preserved for 10 male and 10 female pups per group – where feasible – in F1 offspring not selected for Cohorts on PND22 or shortly thereafter.
Special attention was paid to the organs and tissues of the reproductive system for P or F1 animals.
Selected organ weights were determined in adult animals (P, F1) and in offspring (PND22 or shortly thereafter).
Sperm parameters were determined in all control and high dose male animals in P generation and in F1 generation (Cohort 1A).
Full histopathology examinations were performed on the organs and tissues of adult animals (P, F1 Cohort 1A) in control and high dose groups with special emphasis on sexual organs and tissues.
Reproductive organs were also processed and examined histologically in non-mated and non-pregnant female animals and their mating partners in the low and mid dose groups.
In addition, organs showing macroscopic changes were also processed and examined histologically in adult animals in low or mid dose groups (P, F1 Cohort 1A) and in F1 offspring.
A quantitative evaluation of primordial, small growing (secondary and tertiary) follicles, as well as corpora lutea was performed in F1 Cohort 1A female animals in the control and 180 mg/kg bw/day groups.
The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only.
 
Results
Mortality
Test item related mortality was detected in pregnant female animals (P) at 100 mg/kg bw/day (1/24) and 200/180 mg/kg bw/day (10/24) close to the delivery. Based on necropsy and histopathological findings, the collapse of fluid balance caused the death/ moribund state of these pregnant female animals.
One male animal in the control group of the parental generation (1/24) died because of an individual disease.
There was no test item related mortality in F1 Cohort 1A or F1 Cohort 1B animals in control, 50, 100 or 180 mg/kg bw/day groups (male or female) during the course of study. One female animal at 100 mg/kg bw/day (1/20 Cohort 1B) was found dead on post-natal day 57 due to individual lesion.
 
Clinical observation
Female animals at 100 mg/kg bw/day and male and female animals at 200/180 mg/kg bw/day showed test item related clinical signs referring to systemic toxicity during the course of daily clinical observations.
The main clinical signs appeared at two peak-periods in male animals at 200/180 mg/kg bw/day: before the dose reduction (decreased activity, decreased body tone, diarrhea, piloerection or sanguineous nasal orifice) and at the end of the treatment period (decreased activity and decreased body tone).
In female animals at 100 and 200/180 mg/kg bw/day, the main signs (decreased activity, piloerection, decreased body tone, diarrhea, black discharge around eyes, sanguineous nasal orifice, and sanguineous fore limbs) were detected during the last few days of gestation (G17-21) and first two days of lactation (L0-L1).
Salivation at 100 and 200/180 mg/kg bw/day (male and female) and nuzzling up the bedding material at 200/180 mg/kg bw/day (male and female) were also treatment related sign but were considered to be toxicologically not relevant because of short duration after the test item administration.
Detailed weekly clinical observations also revealed some of these signs in male animals at 200/180 mg/kg bw/day on Day 7 and Day 119 and in female animals at 100 and 200/180 mg/kg bw/day on gestation day 21 and lactation day 0.
Clinical signs related to the test item effect were not detected in male or female animals in F1 Cohort 1A or Cohort 1B generation in 50, 100 or 180 mg/kg bw/day groups at the daily or weekly clinical observations.
 
Body weight and body weight gain
The body weight development was significantly reduced in parental male animals administered with 100 and 200/180 mg/kg bw/day during the entire treatment period and in female animals at 200/180 mg/kg bw/day during gestation period.
Slightly lowered mean body weight of male animals at 50 mg/kg bw/day during the entire treatment period and in female animals at 100 mg/kg bw/day during the gestation period were judged to be toxicologically not relevant because the effect could not be observed in the F1 adult generation and the difference with respect to the control remained less than 10 % both in male and female animals.
The body weight development was reduced in F1 Cohort 1A animals administered with 100 or 180 mg/kg bw/day (male or female).
Similarly, the body weight development was depressed in F1 Cohort 1B animals administered with 180 mg/kg bw/day (male and female).
 
Food consumption
The food consumption was significantly reduced in parental male (pre-mating and post-mating periods) and female animals (pre-mating, gestation and lactation periods) at 100 and 200/180 mg/kg bw/day. The mean daily food consumption of male and female animals was reduced at 100 mg/kg bw/day (Cohort 1A) and at 180 mg/kg bw/day (Cohort 1A and 1B) during the observation period in accordance with the body weight changes.
 
Estrous cycle
The estrous cycle was not adversely affected in all female animals (P, F1A, F1A) at 50, 100 or 200/180 mg/kg bw/day.
 
Delivery data of dams
Lowered mean number of births (total, live and viable pups) as a secondary test item effect on delivery data was observed in dams at 200/180 mg/kg bw/day due to maternal toxicity and high mortality of 200/180 mg/kg bw/day dose treated dams close to the delivery.
 
Reproductive performance
The copulatory performance and fertility of male and female animals (mating and fertilization) were not selectively affected by the treatment with 50, 100 or 200/180 mg/kg bw/day. The lower incidence of dams delivered at 100 and 200/180 mg/kg bw/day was related to the premature death around the day of birth due to severe maternal toxicity of the test item.
 
Urinalysis
The examined urine parameters were not adversely affected in male and female animals at 50, 100 or 200/180 mg/kg bw/day of the parental generation. There were also no test item related changes in the examined urine parameters in F1 Cohort 1A animals (male or female) at 50, 100 or 180 mg/kg bw/day.
 
Clinical pathology
There were no test item related adverse changes in the examined clinical pathology parameters in parental male or female animals at 50, 100 or 200/180 mg/kg bw/day. Elevation of mean activity of alanine aminotransferase at 100 mg/kg bw/day (male) and 200/180 mg/kg bw/day (male and female) as well as lowered albumin and total protein concentrations at 100 or 200/180 mg/kg bw/day (male or female) might be indicative of changed renal or hepatic function as no morphological changes were detected in these organs.
Pathologic alterations were not detected at the evaluation of clinical pathology (urine, hematology and blood coagulation, clinical chemistry) parameters in F1 Cohort 1A male or female animals at 50, 100 or 180 mg/kg bw/day.
 
Thyroid hormones
The serum thyroid hormone (FT3, FT4 and TSH) levels were not adversely influenced in the parental male or female animals, in PND22 F1 offspring and in the F1 Cohort 1A at any dose levels.
 
Necropsy
Necropsy observations revealed changes in the gastro-intestinal tract (stomach, intestines, cecum) in dead pregnant female animals at 100 and 200/180 mg/kg bw/day. Additionally, related signs of these changes and/or agony were detected in the liver, kidneys, spleen, adrenal glands, lungs and on the hairs of these animals.
Macroscopic alterations related to the effect of the test item were not detected in surviving parental male or female animals as well as in F1 Cohort 1A or in F1 Cohort 1B (male or female) animals in all dosing groups at the necropsy.
 
Organ weight
The weights of examined organs were not adversely affected in male or female parental animals at 50, 100 and 200/180 mg/kg bw/day. A test item influence cannot be excluded in development of the lower mean weights of thymus and prostate (male) and higher mean weights of adrenal glands (female) at 200/180 mg/kg bw/day. Cellular damage was not detected at the histopathological examination of these organs and hematology investigations as well as clinical chemistry parameters did not reveal test item related abnormalities. Therefore, weight changes of these organs were judged to be of no toxicological relevance.
The weights of the examined organs were not adversely affected in F1 Cohort 1A or F1 Cohort 1B (male and female) animals at 50, 100 or 180 mg/kg bw/day.
 
Sperm examinations
Sperm examinations did not reveal any test item related influence on the sperm cell morphology and motility, and total sperm count at 200/180 mg/kg bw/day (parental (P) and cohort 1A male animals).
 
Histopathology
Histological examinations revealed of changes in the gastrointestinal tract of dead and moribund female animals (dilatation of stomach, small and large intestines along with large amount of fluid accumulation in the lumen) and exfoliation of mucous membrane – as cause of death –without essential histopathological changes (inflammation, ulceration or other pathological lesions) at 100 and 200/180 mg/kg bw/day. These lesions were not observed in the surviving animals in the mid or high dose groups.
Thymic lymphocyte depletion – indicative of accelerated involution of the organ – in all high dose treated female animals was also considered to be related to test item.
The investigated organs of reproductive system (testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, oviduct, uterus cervix, vagina) were histologically normal and characteristic of the sexually mature organism in all parental (P) animals at 200/180 mg/kg bw/day (including not mated and non-pregnant female animals and their mating partners).
Histological examinations did not reveal pathological alterations in the organs or tissues of F1 Cohort 1A male or female animals at 180 mg/kg bw/day.
 
Splenic Lymphocyte Subpopulation Analysis
There were no test item related changes in the splenic lymphocyte subpopulation in male or female animals in F1 Cohort 1A animals at 50, 100 or 180 mg/kg bw.
 
Offspring
The extra uterine mortality of F1 offspring exceeded the control at 200/180 mg/kg bw/day on post-natal day 0 and between post-natal days 0 and 21. Severe maternal toxicity was considered when evaluating the higher mortality in high dose group.
There were no adverse clinical signs in the F1 offspring from post-natal day 0 to 21. However, the common findings of pups (no milk in the stomach, cold body temperature, percent of dead or missing pups) were observed at a higher incidence than usual at 200/180 mg/kg bw/day corresponding with the inadequate nursing behavior of the respective dams.
The F1 offspring’s development (surface righting reflex, pinna detachment, eye opening) was depressed at 100 and 200/180 mg/kg bw/day most likely due to inadequate nursing behavior of the dams.
The body weight development of the F1 offspring was slightly reduced at 100 and 200/180 mg/kg bw/day.
Specific macroscopic alterations were not found in F1 offspring subjected to gross pathological examination before weaning.
There were no test item related changes in the weights of examined organs (absolute and relative to body and brain weights) in male or female offspring at weaning.
 
In conclusion, Diammonium Peroxodisulphate (APS) caused death of pregnant female animals, clinical signs, reduced body weight and food consumption (male and female), changes in the gastro-intestinal tract – supported by histological findings – in dead pregnant female animals at 200/180 mg/kg bw/day of P generation. The copulatory and fertility ability of male and female animal were not selectively influenced at 200/180 mg/kg bw/day. There was a clearly lower number of dams delivered due to premature death related to severe maternal toxicity  at 200/180 mg/kg bw/day.
At 100 mg/kg bw/day of P generation, mortality of one dam, clinical signs, reduced body weight and food consumption (male and female), macroscopic and histopathological findings (only in dead animal) were observed. The copulatory and fertility ability (male or female animals) was considered normal. One pregnant female did not deliver due to premature death at 100 mg/kg bw/day.
At 50 mg/kg bw/day of P generation, there were no signs of systemic toxicity or influence on the reproductive performance (male or female animals)
In F1 offspring, reduced development (surface righting reflex, pinna detachment, eye opening) and depressed body weight development were observed at 100 and 200/180 mg/kg bw/day.
At 50 mg/kg bw/day of F1 offspring, no test item related effects were detected.
F1 adult (F1 Cohort 1A, Cohort 1B)
Reduced body weight development and food consumption were detected at 100 and 180 mg/kg bw/day in F1 adult animals (male and female). The degree of alterations at 100 mg/kg bw/day were minor and were considered to be toxicologically not relevant.
At 50 mg/kg bw/day of F1 adults, no test item related effects were detected.
Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for systemic toxicity (male and female):       50 mg/kg bw/day
NOAEL for reproductive performance (male):           180 mg/kg bw/day
NOAEL for reproductive performance (female):        50 mg/kg bw/day
NOAEL for F1 Offspring:                                       50 mg/kg bw/day
NOAEL for F1 Adults:                                           100 mg/kg bw/day
 
 
* The high dose of 200 mg/kg bw/day was reduced to 180 mg/kg bw/day on Day 7 – after two days washing out period on Days 5 and 6 – because of clinical signs of toxicity and body weight loss of animals.
 
supporting study (OECD 421)
Diammonium persulfate was tested for oral reproductive/developmental toxicity in a screening test with rats according to OECD guideline 421. The purpose of this study was to obtain initial information on the possible effects of the test item on reproduction and development when administered orally in the diet to Crl:CD (SD)IGS BR rats at doses of 40, 100 and 250 mg/kg bw/day compared to control animals (plain diet only). There were no treatment-related clinical signs of toxicity observed in F0 parents of either sex or in F1 pups at any treatment level. Remarkable clinical signs in the F0 parents and F1 pups were not attributed to treatment with diammonium persulfate, as they occurred sporadically, were of short duration, and did not demonstrate a dose response. No significant changes were observed in male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and in development of the F1 offspring from conception to day 4 postpartum. In conclusion, under the conditions of this study, the NOAEL for male and female toxicity, the NOAEL for male and female fertility performance and the NOAEL for F1 viability and development was 250 mg/kg/day.

Effects on developmental toxicity

Description of key information

Of the Persulfate Category, only diammonium persulfate was tested for developmental toxicity in tests with rats and rabbits according to OECD guideline no. 414.

 

OECD 414, rat

Oral treatment of pregnant Han: Wistar rats from gestation day 5 up to day 19 (the day before Caesarean section) with Diammonium peroxodisulphate (APS) at the dose levels of 10, 30 and 100 mg/kg bw/day did not cause death, or revealed clinical signs and treatment related macroscopic changes at gross pathology. Slightly reduced food intake on GD 5 to 8 and 11 to 14 and slightly lower body weight gain on GD 5 to 8, 17 to 20 and 0 to 20, as well as slightly lower corrected body weight/gain were attributed to the test item at the dose level of 100 mg/kg bw/day. Diammonium peroxodisulphate (APS) did not reveal any adverse effect on the pre- and post-implantation loss, number of implantation, sex distribution, body and placental weight, external, visceral and skeletal development of the fetuses. Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL (maternal toxicity): 30 mg/kg bw/day

NOAEL (developmental toxicity including teratogenicity): 100 mg/kg bw/day

 

OECD 414, rabbit

Oral (gavage) treatment of pregnant New Zealand White rabbits from gestation day 6 up to day 27 (the day before Caesarean section) with Diammonium peroxodisulphate (APS) at the dose level of 100 mg/kg bw/day induced maternal toxicity manifest as initial weight loss and subsequently reduced body weight gain together with reduced food consumption. The NOAEL for maternal toxicity is 30 mg/kg bw/day.

The severity of the maternal toxicity at 100 mg/kg bw/day was considered to impact fetal viability and growth and to slightly delay ossification. This dose of APS did not induce fetal malformations. The NOAEL for developmental toxicity is 30 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
Two studies according to GLP and OECD 414 guideline are available in the rat and in the rabbit. Based on NOAELs obtained, the rabbit being more sensitive than the rat is considered to reflect the worst case.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 414 in the rat

Diammonium peroxodisulphate (APS) was examined for its possible prenatal developmental toxicity in accordance with OECD guideline 414. Groups of 26 sperm-positive female Han: Wistar rats were treated with APS by oral administration daily at three dose levels of 10, 30 and 100 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 26 sperm positive females was included and the animals were given the vehicle water (aqua purificata). The treatment volume was 5 mL/kg bw.

A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation. Diammonium peroxodisulphate (APS) in water was stable at room temperature and in the refrigerator (5 ± 3 °C) for three days at the concentrations of ca. 0.1 and 200 mg/mL. Analytical control of dosing solutions was performed during the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 95 and 104 % of nominal concentrations at both analytical occasions confirming proper dosing.

During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day (GD) 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

In total, on gestation day 20 there were dams with 23, 20, 16 and 16 implantation sites in the control, 10, 30 and 100 mg/kg bw/day group, respectively. None of the females died before scheduled necropsy and there were no test item related clinical signs recorded. No treatment related necropsy findings were observed. Slight, statistically significant decreases of food consumption were indicated in the 100 mg/kg bw/day dose group on GD 5 to 8 and 11 to 14. Slightly lower body weight gain was observed between GD 5 and 8 and 17 and 20. A slightly lower corrected body weight/gain was observed between GD 0 to 20 without a statistical significance.

These differences were in association with the results of the DRF study (Dose Range Finding Prenatal Developmental Toxicity Study with Diammonium Peroxodisulphate (APS) in the Rat by Oral (Gavage) Administration Toxi-Coop study number: 552-410-3000) and were therefore attributed to the test item.

The CH square test indicated a statistical significance in the increase of pre-implantation loss in all test item treated groups. A statistical significant increase of the total intrauterine mortality was observed in the 100 mg/kg bw/day dose group. The mean percentage pre-implantation loss was between the historical control range or slightly above (30 mg/kg bw/day). The mean percentage intrauterine mortality was also between the historical background values. Thus, this was not attributed to treatment. Moreover the mean number of implantations was highest in the 100 mg/kg bw/day dose. There were no dose related increases seen in the post-implantation loss and sex distribution of the fetuses was not influenced by the treatment.

There were no test item related adverse effects on the fetal- and placental weight. There were no test item related external malformations and variations found. The visceral malformations were not attributed to the treatment. The skeletal malformations were found in three fetuses with a statistical significance. However the incidence was low and the type of alterations less severe and partially different. There was no dose related increase seen in external and skeletal variations. Based on these observations the No Observed Adverse Effect Level (NOAELs) were determined as follows: NOAEL (maternal toxicity): 30 mg/kg bw/day, NOAEL (developmental toxicity): 100 mg/kg bw/day, NOAEL (teratogenicity): 100 mg/kg bw/day (high dose).

 

OECD 414 in the rabbit

Diammonium peroxodisulphate (APS) was examined for its possible prenatal developmental toxicity. Groups of 25 (low and mid dose) and 26 (high dose) inseminated New Zealand White rabbits were treated with Diammonium peroxodisulphate (APS) by oral (gavage) administration daily at three dose levels of 10, 30 and 100 mg/kg bw/day respectively from day 6 up to and including day 27 post insemination. A control group of 25 inseminated females was included and the animals were given the vehicle water (aqua purificata). The treatment volume was 2 mL/kg bw.

Diammonium peroxodisulphate (APS) was proved to be stable in water formulations at 0.1 mg/mL and 200 mg/mL concentration levels for three days at room temperature and in the refrigerator (5 ± 3°C) according to the analytical method validation (Study no.: (Toxi-Coop study no. 552-100-3004)) at Toxi-Coop Zrt. The suitability of the chosen vehicle for the test item was analytically proven. Analytical control of dosing solutions was performed during the first and last week of treatment. The mean of the test item concentrations of the test item in the dosing formulations varied in the acceptable range between 96 and 99 % of nominal concentrations at both analytical occasions confirming proper dosing.

During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the does were recorded. The day of insemination was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 28. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed, measured and examined for gross external abnormalities. The placentas were weighed and examined externally.

Fresh visceral examination of each fetus was performed including the determination of the gender.

Head of about 50 % of each litter was removed, fixed in modified Sanomiya solution and washed in 90 % isopropanol. Examination of the heads was done by Wilson's free-hand razor blade method. All skeletons were examined by means of a dissecting microscope after cartilage-bone staining.

There was no test item related mortality, moribund state or abortion observed. In total, on gestation day 28 there were 22, 23, 21 and 20 evaluated litters in the control, 10, 30 and 100 mg/kg bw/day group respectively. There were no test item related clinical signs and pathological macroscopic findings observed.

Treatment with the test item at 100 mg/kg bw/day induced maternal toxicity manifest as an initial weight loss and subsequent reduction in body weight gain (77% between GD 6-28). Corrected body weight and corrected body weight gain clearly reflected the effect at 100 mg/kg bw/day. The reduction in body weight correlated with a reduction in food consumption, observed from the start of the treatment. Treatment with the test item at 30 mg/kg bw/day did not induce maternal toxicity. Variations in weight gain were not statistically significant during the study. Variations in the food consumption were not statistically significant at 30 mg/kg bw/day except for GD 18-21. This did not result in statistically lower body weight gain. There was no effect of 10 mg/kg bw/day on maternal body weight or food consumption.

There was evidence of an increase in early embryonic death/post-implantation loss/total intrauterine mortality and a slightly lower mean number of viable fetuses (without a statistical significance) in the 100 mg/kg bw/day dose group. This outcome was considered to be related to the severity of the maternal toxicity induced.

There was no effect of treatment at 10 or 30 mg/kg bw/day on intrauterine mortality.

Significantly lower fetal weight and crown-rump length were observed in the 100 mg/kg bw/day dose group. These smaller fetuses showed evidence of delayed ossification (e.g. larger or slightly larger anterior fontanelle, reduced or asymmetric ossification of the bones of the digits (including pollex) or small hole in xiphoid cartilage. These effects were considered to be a consequence of the maternal toxicity induced. There was no evidence of treatment-related malformation at 100 mg/kg bw/day. There was no effect of treatment at 10 or 30 mg/kg bw/day on fetal growth or development.

Oral (gavage) treatment of pregnant New Zealand White rabbits from gestation day 6 up to day 27 (the day before Caesarean section) with Diammonium peroxodisulphate (APS) at the dose level of 100 mg/kg bw/day induced maternal toxicity manifest as initial weight loss and subsequently reduced body weight gain together with reduced food consumption. The NOAEL for maternal toxicity is 30 mg/kg bw/day.

The severity of the maternal toxicity at 100 mg/kg bw/day was considered to impact fetal viability and growth and to slightly delay ossification. This dose of Diammonium peroxodisulphate (APS) did not induce fetal malformation.

The NOAEL for developmental toxicity is 30 mg/kg bw/day.

Justification for classification or non-classification

Based on the results obtained, substances of the Persulfate Category were not classified and labelled for reproduction/developmental toxicity according to Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) 2020/1182.

Additional information