Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 15th to 27th, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
preliminary test to define the doses of Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test OECD TG 422.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 15th to 27th, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
According to the protocol of the study, all female pregnant rats should be treated by oral gavage with test item from Day 0 to Day 19 post coitum. The parameters to check in this study should be similar to those evaluated in Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test OECD TG 422 because this study was performed to define the doses to use in main test OECD TG 422 . The focus of the preliminary study should be on the developmental toxicity. However high toxicity was observed during this preliminary test and it was stopped on day 10 post coitum.
GLP compliance:
no
Remarks:
No GLP test but carried out in GLP compliant facility.
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and because there are ample experience and background data on this species and strain.
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females: 50 Sprague Dawley SD virgin female rats, 9 weeks old arrived at the laboratory and they were mated with males rats (11 weeks old) before the beginning of the study.
- Weight at study initiation: between 200-225 g (female rats)
- Housing: before and after mating, the animals were housed no more than 5 of one sex to a cage, in polysulfone cages. Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material was provided as necessary and changed at least 2 times a week. During the mating period (before the beginning of the study), the rats were housed on the basis of 1 male to 1 female in clear polysulfone cages with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily. On the day of allocation (Day 0 post coitum), all females were weighed and allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Each female was identified within the study by ear notch and housed no more than 5 to a cage. The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery to minimise possible environmental effects.
- Diet (e.g. ad libitum): a commercially available laboratory rodent diet was offered ad libitum.
- Water (e.g. ad libitum): drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: an acclimatisation period of approximately 1 week was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22° C ± 2° C
- Humidity (%): 55 % ± 15 %
- Air changes (per hr): from 15 to 20 per hours
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours each day.


Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as it is a possible route of exposure of the test item in man.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1 % aqueous solution of carboxymethylcellulose.
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 5, 10, 15/25 and 50 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
From day 0 post coitum at most till day 10 post coitum.
Frequency of treatment:
Daily.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
group 1: control
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
group 2: low
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
group 3: medium-low
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
group 4: medium-high. Note: the dose for this group was decreased from 250 to 150 mg/kg bw/day from day 5 post coitum due to the high toxicity observed.
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
group 5: high
No. of animals per sex per dose:
6 female pregnat rats per dose ( 6 x 5 = 30 animals included the control group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: preliminary study OECD TG 422 performed to set the doses of the main study OECD TG 422. The doses of the preliminary study was set considering that the substance is classified H302 (acute toxicity cat.4) according to annex VI of the CLP Regulation (EC n.1272/2008).
- Rationale for animal assignment (if not random): on the day of allocation all female rats were weighed and they were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Each female will be identified within the study by ear notch and housed no more than 5 to a cage. The cages will be identified by a label recording the study number, animal numbers and details of treatment. Each group will comprise 6 mated female rats. The rat numbers was formed by the last digits of a computer generated 8 figure animal number (the remaining digits of the animal number will be different for each concurrent study and will serve to ensure unique animal numbering for any study employing computerised data collection). The computerised system used in this study will be the Xybion Path/Tox System, Version 4.2.2.

- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):
Observations and examinations performed and frequency:
MORTALITY
Throughout the study, all animals will be checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure will be followed except that the final check will be carried out at approximately mid-day. This will allow post mortem examinations to be carried out during the working period of that day. Severely debilitated animals will be observed carefully. Animals judged to be in extremis will be killed.

DETAILED CLINICAL OBSERVATIONS
All clinical signs will be recorded for individual animals. Each animal will be observed daily and any clinical signs recorded from allocation until sacrifice.

BODY WEIGHT
All animals were weighed on Days 0, 3, 6, 9 days post coitum when they didn't die before.

Note: Due to the high toxicity observed during the study, many analysis planned in the study protocol were not performed (i.e. Hematology, Coagulation test and Clinical chemistry).
Sacrifice and pathology:
Due to the high toxicity observed the animals were killed at most on day 10 post coitum instead of on day 20 post coitum as planned in the study protocol. The animals were killed under carbon dioxide asphyxiation considering no clinical pathology investigation was necessary.

GROSS PATHOLOGY
From all animals the following organs were dissected free of fat and analysed: adrenal glands,brain, caecum, cervical nodes, duodenum, eyes, forelimbs, harderian glands, head, Ileum, jejunum, kidneys, lacrymal glands, larynx, liver, mesenteric nodes, submandib. nodes, optic nerves, ovaries,parotids, pharynx, pituitary, salivary gland, skin, stomach, thymus, thyroid, tongue, uterus, colon.

Note: Due to the high toxicity observed during the study, many analysis planned in the study protocol were not performed (i.e. pre-implantation loss, post-implantation loss, total implantation loss, sex ratio and examination of foetus).
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The main clinical signs observed, generally starting from the first days of dosing in all treated groups, were cyphosis, decreased activity and piloerection. In addition, in Groups 2 and 3, also emaciated appearance was noted later on. In dead and/or humanely sacrificed animals, also pale appearance and/or prone posture and/or dyspnoea were seen. No relevant clinical signs were observed in control animals.
Mortality:
mortality observed, treatment-related
Description (incidence):
During the treatment of the animals, mortality occurred in all treated groups.
The mortality observed in each group is detailed as follows:
- group 2- low dose group (50 mg/kg bw day): two animals died following dosing at 50 mg/kg/day. The first animal (no. Y0180015) died on Day 8 post coitum and the second one (no. Y0180017) was found dead on Day 9 post coitum. Surviving animals (4 females) were sacrificed for humane reasons the day after interruption of treatment.
- group 3- medium-low dose (100 mg/kg bw/day): two animals died (nos. Y0180027 and Y0180033, on Day 8 and 6 post coitum, respectively) and two were sacrificed for humane reasons (nos. Y0180029 and Y0180031, on Day 9 and 7 post coitum, respectively). Surviving animals (2 females) were sacrificed for humane reasons the day after interruption of treatment.
- group 4- medium-high dose: following dosing at 250/150 mg/kg/day, two animals were found dead (nos. Y0180037 and Y0180045, on Day 6 and 3 post coitum, respectively) and one, no. Y0180039, was sacrificed for humane reasons on Day 3 post coitum. Surviving animals (3 females) were sacrificed for humane reasons the day after interruption of treatment. The interruption of treatment for Group 4 occurred before the interruption of treatment of Groups 3 and 2.
- group 5-high dose (500 mg/kg bw/day): all animals died after dosing at 500 mg/kg/day. Five animals (nos. Y0180049, Y0180051, Y0180055, Y0180057 and Y0180059) died on Day 3 post coitum and the sixth animal (no. Y0180053) was found dead on Day 4 post coitum
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In all treated groups, severe body weight losses and reductions in body weight gain were observed during the observation period starting from Day 3 post coitum, with a dose-related trend. Body weight and body weight gain in control animals were in the normal range for this strain and age of animals.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
500 (Group 5) mg/kg/day were found dead or were sacrificed for humane reasons from Day 3 to Day 11 of study.
- group 2- low dose group (50 mg/kg bw day): the most relevant macroscopic changes observed in most of the treated females (animal nos. Y0180015, Y0180017, Y0180021 and Y0180023) were pale colour of liver and in a single instance (animal no. Y0180023) also enlarged liver. In addition, in one female rat (no. Y0180017), dark colour of kidneys (medulla) and dark areas of glandular region of the stomach were observed.
- group 3- medium-low dose (100 mg/kg bw/day): macroscopically, pale or dark colour of liver, associated or not with soft consistency or enlargement, was observed in all treated females. In addition, in few cases (animal nos. Y0180029 and Y0180031), dark colour of kidneys (medulla) and adrenals, accompanied or not with dark areas of glandular region of the stomach, were noted. In a single case (female no. Y0180027) the animal was almost completely cannibalised.
- group 4- medium-high dose: at post mortem observations, pale and/or soft and/or enlarged and/or swollen liver, associated with dark colour of kidneys (medulla), was noted in all treated females. In addition, in two cases, dark colour of adrenals (animal nos. Y0180043 and Y0180047) and dark areas of glandular region of the stomach (animal nos. Y0180037 and Y0180045) were noted.
- group 5-high dose (500 mg/kg bw/day): the most relevant changes observed at macroscopic examinations in all treated females consisted in pale and soft and/or enlarged liver, associated with dark colour of kidneys (medulla) and dark areas of glandular region of the stomach. In addition, in two cases, enlarged adrenals (animal nos. Y0180051 and Y0180055) were noted.

Female rats treated with the vehicle (Group 1) and killed at termination did not show any macroscopic changes.
Dose descriptor:
LOAEL
Effect level:
ca. 50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
mortality
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Remarks on result:
other: estimated based on the results of preliminary study OECD TG 422.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
liver
kidney
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
LOAEL Tetraphosphorus trisulphide (repeated oral toxicity pregnant female rats treated for 10 days post coitum) = 50 mg/kg bw/day and estimated NOAEL (repeated oral toxicity 28 days rat)= 1 mg/kg bw/day.
Executive summary:

The effects of Tetraphosphorus trisulphide were investigated after oral administration in female rats during pregnancy and on embryo-foetal development. Each treatment group was composed by 6 mated female rats and they received the test item by oral gavage at the dose levels of 0 (group 1:control), 50 (group 2:low), 100 (group 3: medium-low), 250 (group 4: medium-high) and 500 (group 5: high) mg/kg/day at the beginning of the study. All animals were administered during the gestation period, starting from Day 0 post coitum at the dosing volume of 10ml/kg. Due to the severe toxicity observed in the high and medium-high dose groups, the dose level of the medium-high group was reduced from 250 to 150 mg/kg/day from Day 5 post coitum of the first batch of mated females and the treatment of the high dose group was interrupted. No animal of the high dose group survived till the end of the treatment. Despite the reduction of the dose level, mortality was still observed in the medium-high dose group and on Day 6 post coitum of the first batch of mated females also the treatment of this group was interrupted. Surviving animals (3 females) of medium-high dose were sacrificed for humane reasons the day after interruption of treatment.

Severe toxicity was also observed in the medium-low and low dose groups, although at a later stage, and the treatment was stopped on Day 10 post coitum of the first batch of mated females and the study aborted. The treatment of the animals of the control group was stopped on the same day of the low and medium-low dose group animals. At the end of the treatment the surviving animals of medium-low dose and of low dose groups were respectively 2 and 4.

The main clinical signs observed, generally starting from the first days of dosing in all treated groups, were cyphosis, decreased activity and piloerection. In addition, in Groups 2 and 3, also emaciated appearance was noted later on. In dead and/or humanely sacrificed animals, also pale appearance and/or prone posture and/or dyspnoea were seen. No relevant clinical signs were observed in control animals.

In all treated groups, severe body weight losses and reductions in body weight gain were observed during the observation period starting from Day 3 post coitum, with a dose-related trend.

Body weight and body weight gain in control animals were in the normal range for this strain and age of animals.

 The post mortem examination revealed macroscopic changes in liver, kidneys and gastric tract starting from dose of 50 mg/kg bw/day. 

 

Discussion

After 4 days of treatment of the first batch the percentage of deaths in high dose group (500 mg/kg bw/day) was 100 % (6/6) and in the medium-high level group (250 mg/kg bw/ day) was 33 % (2/6). The study was interrupted for high dose group and the dosage was reduced for the medium high-level group from 250 to 150 mg/kg bw/day. Despite of the dose reduction, another death occurred in the medium-high dose group and the study was stopped after 6 days post coitum of the first batch with death mortality of 50 % (3/6). The death in medium-low dose group (100 mg/kg bw/day) and in low dose group (50 mg/kg bw/day) started after day 6 post coitum. Before the interruption of all treatments at day 10 post coitum of first batch, the surviving animals were 4/6 in the low dose group (50 mg/kg bw/day) and 2/6 in the middle-low dose group (100 mg/kg bw/day). The post mortem examination revealed macroscopic changes in liver, kidneys and gastric tract starting from dose of 50 mg/kg bw/day.

 

 

Conclusion

Based on the results of the preliminary study OECD TG 422, the LOAEL repeated dose toxicity oral route for Tetraphosphorus trisulphide was set to 50 mg/kg bw/day. This LOAEL was obtained by preliminary test OECD TG 422 performed on pregnant females rats treated for 10 days post coitum. In the main test OECD TG 422 the male and female rats should be treated respectively for about 28 days and for approximately 63 days (at least about 50 days). However, considering the high toxicity observed in preliminary test OECD TG 422 and according to animal welfare legislation, the complete OECD TG 422 on Tetraphosphorus trisulphide was not performed and on precautionary principle NOAEL (repeated oral toxicity 28 days rat)= 1 mg/kg bw/day was chosen to calculate DNEL value. This approach is conservative because, starting from NOAEL =1 mg/kg bw/day, the DNEL value obtained for general population oral route regarding long term exposure is 1.6 μg/kg bw/day, equal to TTC (Threshold of Toxicological Concern) approach 1.5 μg/kg bw/day applied to carcinogenic substances.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
According to the protocol of the study, all female pregnant rats should be treated by oral gavage with test item from Day 0 to Day 19 post coitum. The parameters to check in this study should be similar to those evaluated in Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test OECD TG 422 because this study was performed to define the doses to use in main test OECD TG 422 . The focus of the preliminary study should be on the developmental toxicity. However high toxicity was observed during this preliminary test and it was stopped on day 10 post coitum.
GLP compliance:
no
Remarks:
No GLP test but carried out in GLP compliant facility.
Test type:
other: preliminary test to define the doses of Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test OECD TG 422.
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females: 50 Sprague Dawley SD virgin female rats, 9 weeks old arrived at the laboratory and they were mated with males rats (11 weeks old) before the beginning of the study.
- Weight at study initiation: between 200-225 g (female rats)
- Housing: before and after mating, the animals were housed no more than 5 of one sex to a cage, in polysulfone cages. Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material was provided as necessary and changed at least 2 times a week. During the mating period (before the beginning of the study), the rats were housed on the basis of 1 male to 1 female in clear polysulfone cages with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily. On the day of allocation (Day 0 post coitum), all females were weighed and allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Each female was identified within the study by ear notch and housed no more than 5 to a cage. The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery to minimise possible environmental effects.
- Diet (e.g. ad libitum): a commercially available laboratory rodent diet was offered ad libitum.
- Water (e.g. ad libitum): drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: an acclimatisation period of approximately 1 week was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22° C ± 2° C
- Humidity (%): 55 % ± 15 %
- Air changes (per hr): from 15 to 20 per hours
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours each day.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1 % aqueous solution of carboxymethylcellulose.
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 5, 10, 15/25 and 50 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg

DOSAGE PREPARATION (if unusual): the test item was suspended/dissolved in the vehicle. The formulation was prepared daily.
Doses:
0 (group 1: control), 50 (group 2: low), 100 (group 3:medium-low), 250/150 (group 4: medium-high), 500 (group 5: high) mg/kg/day.
No. of animals per sex per dose:
6 female pregnat rats per dose ( 6 x 5 = 30 animals included the control group)
Control animals:
yes
Details on study design:
- Duration of observation period following administration:10 days post coitum of the first batch of mated females. Due to the high toxicity observed during the study, the test was stopped at 10 day post coitum before the period planned in the study protocol (19 days post coitum)
- Frequency of observations and weighing: daily during all the in vivo phase. All animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays, a similar procedure was followed except that the final check was carried out at approximately mid-day. All clinical signs were recorded for individual animals. Each animal was observed daily and any clinical signs recorded from allocation until sacrifice. All animals were weighed on Days 0, 3, 6, 9 days post coitum when they didn't die before.
- Necropsy of survivors performed: yes
- Other examinations performed: the organ were macroscopically evaluated: thyroid, thymus, spleen, kidneys and liver were dissected free of fat and weighed.
Note: Due to the high toxicity observed during the study, many analysis planned in the study protocol were not performed (i.e.Hematology, Coagulation test, Clinical chemistry,litter data, sex ratio and the examination of foetus).The animals were killed under carbon dioxide asphyxiation considering no clinical pathology investigation was necessary.

Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 50 - <= 300 mg/kg bw
Based on:
test mat.
Remarks on result:
other: See remarks
Remarks:
LD50value was extrapolated based on the results of the preliminary study OECD TG 422 on pregnant rats.
Mortality:
During the treatment of the animals, mortality occurred in all treated groups.
The mortality observed in each group is detailed as follows:
- group 2- low dose group (50 mg/kg bw day): two animals died following dosing at 50 mg/kg/day. The first animal (no. Y0180015) died on Day 8 post coitum and the second one (no. Y0180017) was found dead on Day 9 post coitum. Surviving animals (4 females) were sacrificed for humane reasons the day after interruption of treatment.
- group 3- medium-low dose (100 mg/kg bw/day): two animals died (nos. Y0180027 and Y0180033, on Day 8 and 6 post coitum, respectively) and two were sacrificed for humane reasons (nos. Y0180029 and Y0180031, on Day 9 and 7 post coitum, respectively). Surviving animals (2 females) were sacrificed for humane reasons the day after interruption of treatment.
- group 4- medium-high dose: following dosing at 250/150 mg/kg/day, two animals were found dead (nos. Y0180037 and Y0180045, on Day 6 and 3 post coitum, respectively) and one, no. Y0180039, was sacrificed for humane reasons on Day 3 post coitum. Surviving animals (3 females) were sacrificed for humane reasons the day after interruption of treatment. The interruption of treatment for Group 4 occurred before the interruption of treatment of Groups 3 and 2.
- group 5-high dose (500 mg/kg bw/day): all animals died after dosing at 500 mg/kg/day. Five animals (nos. Y0180049, Y0180051, Y0180055, Y0180057 and Y0180059) died on Day 3 post coitum and the sixth animal (no. Y0180053) was found dead on Day 4 post coitum.
Clinical signs:
The main clinical signs observed, generally starting from the first days of dosing in all treated groups, were cyphosis, decreased activity and piloerection. In addition, in Groups 2 and 3, also emaciated appearance was noted later on. In dead and/or humanely sacrificed animals, also pale appearance and/or prone posture and/or dyspnoea were seen. No relevant clinical signs were observed in control animals.
Body weight:
In all treated groups, severe body weight losses and reductions in body weight gain were observed during the observation period starting from Day 3 post coitum, with a dose-related trend. Body weight and body weight gain in control animals were in the normal range for this strain and age of animals.
Gross pathology:
500 (Group 5) mg/kg/day were found dead or were sacrificed for humane reasons from Day 3 to Day 11 of study.
- group 2- low dose group (50 mg/kg bw day): the most relevant macroscopic changes observed in most of the treated females (animal nos. Y0180015, Y0180017, Y0180021 and Y0180023) were pale colour of liver and in a single instance (animal no. Y0180023) also enlarged liver. In addition, in one female rat (no. Y0180017), dark colour of kidneys (medulla) and dark areas of glandular region of the stomach were observed.
- group 3- medium-low dose (100 mg/kg bw/day): macroscopically, pale or dark colour of liver, associated or not with soft consistency or enlargement, was observed in all treated females. In addition, in few cases (animal nos. Y0180029 and Y0180031), dark colour of kidneys (medulla) and adrenals, accompanied or not with dark areas of glandular region of the stomach, were noted. In a single case (female no. Y0180027) the animal was almost completely cannibalised.
- group 4- medium-high dose: at post mortem observations, pale and/or soft and/or enlarged and/or swollen liver, associated with dark colour of kidneys (medulla), was noted in all treated females. In addition, in two cases, dark colour of adrenals (animal nos. Y0180043 and Y0180047) and dark areas of glandular region of the stomach (animal nos. Y0180037 and Y0180045) were noted.
- group 5-high dose (500 mg/kg bw/day):  the most relevant changes observed at macroscopic examinations in all treated females consisted in pale and soft and/or enlarged liver, associated with dark colour of kidneys (medulla) and dark areas of glandular region of the stomach. In addition, in two cases, enlarged adrenals (animal nos. Y0180051 and Y0180055) were noted.
 
Female rats treated with the vehicle (Group 1) and killed at termination did not show any macroscopic changes.
Other findings:
Due to the high toxicity observed during the study many analysis planned in the study protocol were not performed (i.e.Hematology, Coagulation test, Clinical chemistry,litter data and sex ratio and examination of foetus).The animals were killed under carbon dioxide asphyxiation considering no clinical pathology investigation was necessary.

Applicant's summary and conclusion

Interpretation of results:
other: classified H301 (Acute oral tox cat.3) according to criteria of the CLP Regulation (EC n.1272/2008)
Conclusions:
Extrapolated LD50 oral rat between 50 and 300 mg/kg bw/day.
Executive summary:

The effects of Tetraphosphorus trisulphide were investigated after oral administration in female rats during pregnancy and on embryo-foetal development. Each treatment group was composed by 6 mated female rats and they received the test item by oral gavage at the dose levels of 0 (group 1:control), 50 (group 2:low), 100 (group 3: medium-low), 250 (group 4: medium-high) and 500 (group 5: high) mg/kg/day at the beginning of the study. All animals were administered during the gestation period, starting from Day 0 post coitum at the dosing volume of 10ml/kg. Due to the severe toxicity observed in the high and medium-high dose groups, the dose level of the medium-high group was reduced from 250 to 150 mg/kg/day from Day 5 post coitum of the first batch of mated females and the treatment of the high dose group was interrupted. No animal of the high dose group survived till the end of the treatment. Despite the reduction of the dose level, mortality was still observed in the medium-high dose group and on Day 6 post coitum of the first batch of mated females also the treatment of this group was interrupted. Surviving animals (3 females) of medium-high dose were sacrificed for humane reasons the day after interruption of treatment.

Severe toxicity was also observed in the medium-low and low dose groups, although at a later stage, and the treatment was stopped on Day 10 post coitum of the first batch of mated females and the study aborted. The treatment of the animals of the control group was stopped on the same day of the low and medium-low dose group animals. At the end of the treatment the surviving animals of medium-low dose and of low dose groups were respectively 2 and 4.

The main clinical signs observed, generally starting from the first days of dosing in all treated groups, were cyphosis, decreased activity and piloerection. In addition, in Groups 2 and 3, also emaciated appearance was noted later on. In dead and/or humanely sacrificed animals, also pale appearance and/or prone posture and/or dyspnoea were seen. No relevant clinical signs were observed in control animals.

In all treated groups, severe body weight losses and reductions in body weight gain were observed during the observation period starting from Day 3 post coitum, with a dose-related trend.

Body weight and body weight gain in control animals were in the normal range for this strain and age of animals.

 The post mortem examination revealed macroscopic changes in liver, kidneys and gastric tract starting from dose of 50 mg/kg bw/day. 

 

Discussion

Even though the test item was administered at repeated dose to pregnant rats in this preliminary OECD TG 422 study,  some information regarding LD50 oral of Tetraphosphorus trisulphide can be extrapolated. After 4 days of treatment of the first batch the percentage of deaths in high dose group (500 mg/kg bw/day) was 100 % (6/6) and in the medium-high level group (250 mg/kg bw/ day) was 33 % (2/6). The study was interrupted for high dose group and the dosage was reduced for the medium high-level group from 250 to 150 mg/kg bw/day. Despite of the dose reduction, another death occurred in the medium-high dose group and the study was stopped after 6 days post coitum of the first batch with death mortality of 50 % (3/6). The death in medium-low dose group (100 mg/kg bw/day) and in low dose group (50 mg/kg bw/day) started after day 6 post coitum. Before the interruption of all treatments at day 10 post coitum of first batch, the surviving animals were 4/6 in the low dose group (50 mg/kg bw/day) and 2/6 in the middle-low dose group (100 mg/kg bw/day). 

 

Conclusion

Based on the results of the preliminary study OECD TG 422, LD50 oral rat was reasonable set between 50 and 300 mg/ kg bw/day.