[No public or meaningful name is available]

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From May 25th to June 18th, 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

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:

All animals were administered during the gestation period, starting from Day 6 through Day 19 post coitum. The parameters observed are similar to those evaluated in Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test OECD TG 422 and this study was performed to define the doses to use in main test OECD TG 422 focusing the attention on the developmental toxicity.

GLP compliance:

no

Remarks:

information obtanied from a preliminary OECD 422 study

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

Test material information

Constituent 1

Reference substance name:

Reaction mass of dodecane-1-thiol and tridodecyl trithiophosphite

Molecular formula:

Not applicable - Multiconstituent substance

IUPAC Name:

Reaction mass of dodecane-1-thiol and tridodecyl trithiophosphite

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: at least 11 weeks old.
- Weight at study initiation: at least 340 g.
- Housing: during the pre-pairing period 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, 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. Overnight fasting conditions was respected before the blood sample on Day 20 post coitum.
- 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): 15 to 20 air changes per hour.
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours each day.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 25, 75, 225 and 500 mg/ml
- Amount of vehicle (if gavage): 2 ml/kg. Control animals received the vehicle alone at the same dose volume .The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

DOSAGE PREPARATION: the formulation was prepared daily.

Doses:

0, 50, 150, 450 and 1000 mg/kg bw/day. Administration by gavage once a day from Day 6 through Day 19 post coitum.

No. of animals per sex per dose:

6 females rat for each group ( 6 x 5 = 30 animals included the control group)

Control animals:

yes

Details on study design:

- Duration of observation period following administration: body weight and daily clinical signs were recorded during all the in vivo phase (till day 20 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, 12, 15, 18 and 20 post coitum.
- Necropsy of survivors performed: yes, performed on day 20 post coitum. All animals were killed by exsanguination under isofluorane anaesthesia on Day 20 post coitum and necropsied as detailed below. All animals were subjected to necropsy, super-vised by a pathologist. All foetuses were sacrificed by intraperitoneal injection of Sodium Thiopental followed by hypothermia.
- Other examinations performed:
# ORGAN WEIGHTS: thyroid, thymus, spleen, kidneys and liver were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.
# HAEMATOLOGY: the following parameters were analysed: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count (i.e. Neutrophils, Lymphocites, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelets.
# COAGULATION: Prothrombin time was defined for each animal.
# CLINICAL CHEMISTRY: the following parameters were analysed: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Inorganic phosphorus, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/GRatio, Sodium, Potassium, Calcium, Chloride.
# LITTER DATA AND SEX RATIOS: after necrosis the ovaries and uteri were examinated to determine: gravid uterine weight, number of corpora lutea, number of implantation sites, number, sex and weight of all live foetuses, number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing), number of intra-uterine deaths, gross evaluation of placentae, intra-uterine (early and late resorptions).
# EXAMINATION OF FOETUSES: all live foetuses were examined externally and all anomalies were registered.

CALCULATION:
Group mean values for body weight of pregnant females, gravid uterus weight, absolute weight gain (terminal body weight minus body weight at Day 0 post coitum minus gravid uterus), litter size, intra-uterine deaths, corpora lutea count, total implantation loss and pre-and post-implantation loss were calculated.
Data from non-pregnant animals (including those with total resorption) were not included in group mean calculations of maternal body weight.
PRE IMPL. LOSS % : (no of corpora lutea - no. of implantations ) / no. of corpora lutea X 100
POST IMPL. LOSS%: (no of implantations - no. of live foetuses ) / no. of implantations X 100
TOTAL IMPL. LOSS %: (no of corpora lutea - no. of live foetuses ) / no. of corpora lutea X 100
All derived values (e.g., means, percentages, ratios) were first calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.

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

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: See remarks

Remarks:

LD50 > 2000 mg/kg derives from WoE Approach based on the result of the preliminary OECD TG 422 study combining with the result of the main study OECD TG 422.

Mortality:

No animals died during the study. One female of the low dose dose group (no. Y0170015) and one of the mid-low dose group (no. Y0170033) were not pregnant. In addition, one low dose group female (no. Y0170021) had unilateral implantation. The number of females with live foetuses on gestation Day 20 was: 6 in each of the control, mid-high and high dose groups and 5 in each of the low and mid-low groups.

Clinical signs:

other: No relevant signs of toxicity were observed during the dosing period. The clinical signs observed were: salivation in two females (nos. Y0170051 and Y0170053) of the high dose group and observed for one day only. In addition, scabs and/or hairloss in diff

Gross pathology:

No treatment-related absolute and relative organ weight changes (included gravid uterus) were seen. The minimal statistically significant decrease in relative liver mean weight (- 8%) seen in the mid-low dose females at final sacrifice was considered incidental as unrelated to the dose and as all individual values were within the physiological range of variability.

Other findings:

- Other observations:
# HAEMATOLOGY: No changes of toxicological relevance were observed in haematological parameters.One female dosed at 1000 mg/kg/day (no. Y0170057) showed slight decrease of erythrocytes, haemoglobin and haematocrit (approximately 18% below mean control data). Due to the low incidence, this change was considered incidental.
# COAGULATION: No changes were recorded in the coagulation parameter investigated.
# CLINICAL CHEMISTRY: Fluctuations in some liver parameters were recorded in individual animals of each treated group. Due to the absence of dose-relation, the lack of association between findings and the minimal incidence, the fluctuations were not conclusively attributed to treatment. Compared with mean control data, sporadic increases of some liver parameters were re-corded, such as: alkaline phosphatase in female nos. Y0170019 (50 mg/kg/day, 2.3 fold), Y0170033 (150 mg/kg/day, 2.0 fold), Y0170049 (1000 mg/kg/day, 2.3 fold) and Y017055 (1000 mg/kg/day, 2.9 fold), alanine aminotransferase in female no. Y0170059 (1000 mg/kg/day, 2.0 fold), aspartate aminotransferase in animal no. Y0170023 (50 mg/kg/day, 1.8 fold) and bilirubin in animal nos. Y0170019 (50 mg/kg/day, 5.0 fold), Y0170031 (150 mg/kg/day, 4.2 fold), Y0170045 (450 mg/kg/day, 6.7 fold), and Y0170049 (1000 mg/kg/day, 5.0 fold). Due to the absence of dose-relation, the lack of association between findings and the minimal incidence, the above findings were not conclusively attributed to treatment.
# LITTER DATA AND SEX RATIOS: Litter data, mean foetal weight and sex ratios were not affected by treatment.
# EXTERNAL EXAMINATION OF FOETUSES: At the external examination, no abnormalities were detected in foetuses of treated females, with the exception of one small foetus in the mid-low and mid-high dose group each, which were considered incidental and not related to treatment.

Applicant's summary and conclusion

Interpretation of results:

other: not classified for acute oral toxicity according to the CLP Regulation (EC n.1272/2008)

Conclusions:

LD50 rat oral route > 2000 mg/kg bw and no classification for acute oral toxicity is required according to the CLP Regulation (EC n.1272/2008).

Executive summary:

The effects of the substance were investigated in the Sprague Dawley rat, after oral administration from Day 6 to Day 19 of gestation. Each treatment group comprised 6 mated female rats and received the test item orally at the dose levels of 50, 150, 450 and 1000 mg/kg/day. No animals died during the study. Neither clinical signs nor significant signs of reaction to treatment were noted in treated females. No differences were noted in body weight, gravid uterus weight, organ weights, litter data and macroscopic observation of treated females, when compared to controls. Fluctuation and changes observed at the clinical pathology analysis were considered incidental or not conclusively attributed to treatment. Considering the purpose of this preliminary study, some post-mortem specific examination regarding the prenatal developmental toxicity were performed but as overall assessment it can be concluded that the test item did not induce toxic effects in dams or foetuses up to and including the high dose tested of 1000 mg/kg/day. As reported in ECHA guidance R7a (July 2017) if in a subacute oral toxicity study NOAEL is 1000 mg/kg bw/day is possible to assume than LD 50 > 2000 mg/kg bw. This hypothesis is supported in this specific case also considering that the rats of this subacute test were pregnant and more sensitive than the standard requirement for animals in acute toxicity tests.

In conclusion, LD50 oral rat for the substance is reasonable higher than 2000 mg/kg bw.