Registration Dossier

Administrative data

Description of key information

Acute oral toxicity: LD50 (male/female): >2000 mg/kg bw (OECD 423/GLP)

Acute inhalational toxicity: LD50 (male/female): > 5 mg/L (4 hrs; nose only; OECD 403/GLP)

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 May 2017 - 25 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Willing New Material Technology Co.,Ltd.; 23161211201
- Expiration date of the lot/batch: 10 December 2017
- Purity: 97%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 ºC)


Species:
rat
Strain:
other: Crl:WI Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks
- Weight at study initiation: 180 - 202 g
- Fasting period before study: Yes, night before
- Housing: Type II polypropylene/polycarbonate cages; Lignocel 3/4-S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH + CO.KG (D-73494 Rosenberg, Germany). Arbocel crinklets natural nesting produced by J. Rettenmaier & Söhne GmbH + CO.KG (D-73494 Rosenberg, Germany).
- Diet & water: Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany (Batch number: 285 17890, expiry date: 31 August 2017), ad libitum, and tap water from the municipal supply, as for human consumption from a 500 ml bottle, ad libitum. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Acclimation period: 6 and 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 – 25.0 °C
- Humidity (%): 31 – 58 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.



Route of administration:
oral: gavage
Vehicle:
other: 1 % methyl cellulose
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 200 mg/mL
- Lot/batch no. (if required): 5115851

The test item was freshly formulated at a concentration of 200 mg/mL in the 1 % methyl cellulose (dispensary code: S11129), in the Pharmacy of CiToxLAB Hungary Ltd. on the day of administration. The formulation container was magnetic stirred continuously up to the end of dose administration procedures
Doses:
Group 1: 2000 mg/kg bw
Group 2: 2000 mg/kg bw
No. of animals per sex per dose:
3 females
Control animals:
no
Details on study design:
Clinical Observations
Clinical observations were performed on all animals at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. Individual observations were performed on the skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Body Weight Measurement
The body weight was recorded on the day before treatment (Day -1), on the day of the treatment (Day 0), weekly thereafter (Day 7) and at necropsy (Day 14).

Necropsy
Macroscopic examination was performed on all animals. The animals were sacrificed by exsanguination under pentobarbital anaesthesia (Release; Lot No.: 106 075, Expiry date: July 2018, produced by: Wirtschaftsgenossenschaft deutscher Tierärzte eG, Siemensstr. 14, 30827 Garbsen, Germany). After examination of the external appearance, the cranial, thoracic and the abdominal cavities were opened and the organs and the tissues were observed.
Statistics:
The method used was not intended to allow the calculation of a precise LD50 value.
The method used was not intended to allow the calculation of a precise LD50 value. The test item was ranked into categories of Globally Harmonized Classification System (GHS (rev. 6) 2015). Clinical signs, body weight, body weight gain and gross macroscopic data were tabulated.
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Mortality:
TiBTD did not cause mortality at a dose level of 2000 mg/kg bw in any animal.
Clinical signs:
All animals were symptom-free during the observation period at a dose level of 2000 mg/kg bw.
Body weight:
Body weight gains of TiBTD treated animals during the study showed no indication of a test item-related effect
Gross pathology:
There was no evidence of the macroscopic observations in animals dosed at 2000 mg/kg bw and subjected to the necropsy on Day 14.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, the acute oral LD50 value of the test item TiBTD was found to be above 2000 mg/kg bw in female Crl:WI Wistar rats.
Executive summary:

In an acute oral toxicity study (17/053-001P), Crl:WI Wistar female rats (3/dose) were given TiBTD (97%) in 1% methyl cellulose at doses of 2000 mg/kg bw and observed for 14 days.

LD50 (female): was >2000 mg/kg bw.

Initially, 3 females were treated at a dose level of 2000 mg/kg bw. As no mortality was observed, a confirmatory group of 3 females was treated at the same dose level. TiBTD did not cause mortality at a dose level of 2000 mg/kg bw in any animal. All animals were symptom-free during the observation period at a dose level of 2000 mg/kg bw. Body weight gains of TiBTD treated animals during the study showed no indication of a test item-related effect. There was no evidence of the macroscopic observations in animals dosed at 2000 mg/kg bw and subjected to the necropsy on Day 14.

This acute oral study is classified as acceptable. It does satisfy the guideline requirement for an acute oral study (OECD 423) in the rat.  

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
2 000 mg/kg bw
Quality of whole database:
There is one key study available and it an OECD 423/GLP study. The quality of the database is high.

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 September 2017 - 15 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
GLP compliance:
yes (incl. certificate)
Test type:
traditional method
Limit test:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Willing; 23170911201
- Expiration date of the lot/batch: 10 September 2018
- Purity: 97%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 ºC), protected from humidity

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
As the test item was not suitable for atmosphere generation in the form supplied, a series of formulations using acetone (AnalaR NORMAPUR ACS, Reag. Ph. Eur., Batch: 15J060514, Expiry: 31 October 2020) were tested. The 50 w/w % formulation allowed the most appropriate test atmosphere parameters and this was used in the animal exposures.

Species:
rat
Strain:
other: Wistar Crl:WI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Model and Services, Germany GmbH (Sandhofer Weg 7, D-97633, Sulzfeld, Germany).
- Age at study initiation: Sighting exposure - 13 weeks old; Main study - 8 weeks old
- Weight at study initiation: Sighting exposure: 444 g (male) and 236 g (female); Main study: 332-366 g (males) and 215-237 g (females).
- Fasting period before study:
- Housing: Group caging (5 animals, by sex, per cage), individual caging during the sighting study. Polycarbonate solid floor cages (type II or III) with stainless steel mesh lids. Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co. KG (Holzmühle 1, D-73494 Rosenberg, Germany), was used. The quality of the bedding material was guaranteed by the supplier. Nest building material (ARBOCEL crinklets natural (produced by J. Rettenmaier & Söhne GmbH & Co.KG, Germany)) was also added to the cages.
- Diet: Ssniff SM R/M “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” (ssniff Spezialdiäten GmbH, D-59494 Soest Germany) ad libitum
- Water: Tap water from the municipal supply ad libitum
- Acclimation period: Animals were acclimated to laboratory conditions for 41 days (sighting study) or 6 days (main study) prior to involvement in the study. Animals were also acclimatised to the test apparatus (restraint procedures) for a short period prior to testing in order to lessen the stress during exposure.

ENVIRONMENTAL CONDITIONS
- Temperature: 19.0 – 25.0 °C
- Humidity: 36 – 61 %
- Air changes: At least 15 air exchanges per hour.
- Photoperiod: 12 hours of continuous artificial light in each twenty-four-hour period (from 6.00 a.m. to 6.00 p.m.)
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: acetone
Mass median aerodynamic diameter (MMAD):
> 1.51 - < 1.55 µm
Geometric standard deviation (GSD):
> 2 - < 2.05
Remark on MMAD/GSD:
Sighting study:
MMAD: 1.55 µm
GSD: 2.05
Inhalable fraction (<4 µm): 90.5 %

Main study:
MMAD: 1.51 µm
GSD: 2.00
Inhalable fraction (<4 µm): 92.0 %
Details on inhalation exposure:
Inhalation Exposure

Technical Trials
Prior to animal exposures, test material atmospheres were generated within the exposure chamber. During these technical trials, air-flow settings, test material input rates and test item formulation as supplied by the Sponsor were varied to achieve the required aerosol concentration of particles with a mass median aerodynamic diameter (MMAD) between 1 to 4 µm and a geometric standard deviation (GSD) in the range of 1.5 to 3.0. Measurements of aerodynamic particle size were performed from the animal’s breathing zone using a cascade impactor.

Atmosphere generation
The test item formulation was aerosolized using a stainless steel concentric jet nebulizer (TSE Systems GmbH, Bad Homburg, Germany) located at the top of the exposure chamber. The rate of test item usage was controlled by a syringe pump.Compressed air was supplied by means of an oil-free compressor and passed through a suitable filter system prior to introduction to the nebuliser.

Animal exposure system
The animals were exposed, nose-only, to an atmosphere of the test item using a TSE Rodent Exposure System (TSE Systems GmbH, Bad Homburg, Germany). This system comprises of two concentric anodised aluminium chambers and a computer control system incorporating pressure detectors and mass flow controllers. The exposure unit was placed in closed hood in order to avoid cross-contamination and contamination of the laboratory environment. Fresh aerosol from the generation system was constantly supplied to the inner plenum (distribution chamber) of the exposure system from where, under positive pressure, it was distributed to the individual exposure ports. The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animal’s nostrils to enter the exposure port. After passing through the animal’s breathing zone, used aerosol entered the outer cylinder from where it was exhausted through a suitable filter system. Atmosphere generation was therefore dynamic. Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and constant flow of fresh aerosol to each exposure port (breathing zone). The flow of air through each port was at least 0.5 L/min. This flow rate was considered adequate to minimize re-breathing of the test atmosphere as it is approximately twice the respiratory minute volume of a rat. Homogeneity of the test atmosphere within the test chamber and amongst the exposure ports was not specifically determined during this study. However, considerably larger chambers of this design have been fully validated and have shown to produce evenly distributed atmospheres in the animals’ breathing zones.

Sighting Exposure
Sighting exposure was performed in order to estimate the item’s inhalation toxicity, identify sex differences in susceptibility and assist in selecting exposure concentration levels for the main study.

Exposure procedure
Each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber. Only the nose of each animal was exposed to the test atmosphere.
Following an equilibration period of at least the theoretical chamber equilibration time (T99), each group of rats was exposed to an atmosphere of the test material for a period of 4 hours.
No control animals were used in the study.

Exposure Monitoring

Test atmosphere concentrations
The test atmosphere was sampled at regular intervals during the exposure period. Samples were taken from an unoccupied exposure port (representing the animal’s breathing zone) by pulling a suitable volume of test atmosphere through weighed GF10 glass fibre filters (Whatman®, Whatman GmbH, Germany, Ref./Lot no.: A10058746). The difference in the pre and post sampling (dried) weights, divided by the volume of atmosphere sampled, was equal to the actual achieved test atmosphere concentration. Filter samples (17) were collected at the breathing zone (approximately every 10-20 minutes) during each 4-hour exposure period and analysed. The nominal concentration was calculated by dividing the mass of test material disseminated into the chamber by the total volume of air that flow through the chamber during the same period.

Particle size analysis
The particle size of the test atmosphere was determined three times during the exposure period using a 7-stage impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Such devices employ an inertial separation technique to isolate particles in the discrete aerodynamic size ranges. Samples were taken from an unoccupied exposure port (representing the animal’s breathing zone).
The collection substrates and the backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by this difference.
The total amount collected for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than < 0.550, 0.550, 0.960, 1.550, 2.105, 3.555, 6.655 and 10.550 µm was calculated.
From these data, using the software supplied with the impactor (TSE Systems GmbH, Bad Homburg, Germany), the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated. In addition, the proportion (%) of aerosol less than 4 µm (considered to be the inhalable portion) was determined.

Chamber environmental conditions
The following variables were monitored continuously and recorded at regular intervals during each exposure period by a validated monitoring system integrated into the exposure system:
•Chamber airflow rates
•Test atmosphere temperature
•Test atmosphere relative humidity
•Test atmosphere carbon dioxide concentration
•Test atmosphere oxygen concentration
Summaries of the data are presented in Appendix 3.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Sighting exposure:
Target Concentration: 5 mg/L
Mean Achieved Concentration: 4.94 ± 0.33 mg/L

Main study exposure:
Target Concentration: 5 mg/L
Mean Achieved Concentration: 4.96 ± 0.32 mg/L
No. of animals per sex per dose:
Sighting study: 1 male and 1 female

Main study: 5 males and 5 females
Control animals:
no
Remarks:
No control group was exposed in this study because the effects of the vehicle are well known.
Details on study design:
- Duration of observation period following administration: Animals were checked hourly during exposure, 1 hour after exposure and twice daily (early and late in the working day) during the 14-day observation period for morbidity and/or mortality.

- Frequency of observations and weighing: Individual body weights were recorded prior to treatment on the day of exposure (on Day 0) and on Days 1, 3, 7 and 14.

- Necropsy of survivors performed: Yes. At the end of the 14-day observation period, the surviving animals were sacrificed by exsanguination under anaesthesia (intra-peritoneal injection of pentobarbital solution (Euthanimal 40%; Lot No.: 1609291-03, Expiry Date: 31 October 2019, Produced by: Alfasan, Kuipersweg 9 Woerden, The Neetherlands) and gross macroscopic examination was performed. All rats were subject to a gross necropsy which included a detailed examination of the abdominal and thoracic cavities. Special attention was given to the respiratory tract for macroscopic signs of irritancy or local toxicity.

- Other examinations performed: All animals were observed for clinical signs at hourly intervals during exposure whilst the animals were still restrained. Following exposure, clinical observations were performed twice on the day of exposure (following removal from the restrainer and approximately one hour after completion of the exposure) and subsequently once daily for 14 days. Observations included changes in the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somato-motor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.



Statistics:
Only a limit test was performed, the four-hour inhalation LC50 was not calculated.
Preliminary study:
Mortality
No deaths occurred during the study (Appendix 4).

Clinical Observations
In the sighting group, red-brown staining or fur staining by the test item were commonly recorded on the day of the exposure or on Day 1 which were considered to be related to the restraint and exposure procedures but not to be toxicologically significant. Laboured respiration (slight) and incoordination in both animal and decreased activity in male rat were recorded on the day of exposure. The animals were symptom free from Day 1 (Appendix 5).

Body weight
Slight (0.9 – 1.7 %) body weight loss was shown between Day 0 and Day 7. The bodyweight gain returned to the normal range by Day 14 (Appendix 6).
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5 mg/L air (analytical)
Exp. duration:
4 h
Remarks on result:
not determinable due to absence of adverse toxic effects
Mortality:
No deaths occurred during the study (Appendix 4).
Clinical signs:
Laboured respiration (slight) was recorded in 1/10 animals on the day of exposure. The animals were symptom free from Day 1 (Appendix 5).
Body weight:
Slight (0.6 %) body weight loss was shown in one male rat between Day 0 and Day 7. In the observation period, normal body weight gain was observed (Appendix 6).
Gross pathology:
Following a single four-hour nose-only exposure of TiBTD to CRL: (WI) Wistar rats dosed at 4.96 mg/L, for the Main study groups and with a 14 day of observation period, there was no external or internal findings could be detected during the necropsy. Individual necropsy findings and the pathology report are presented in Appendices 7 and 8, respectively.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, no mortality occurred in a group of 10 rats (main study) when exposed to a test atmosphere concentration of 4.96 mg/L for 4 hours. The acute inhalation median lethal concentration (LC50) of TiBTD in Wistar Crl:WI rats was therefore considered to be > 5 mg/L.
Executive summary:

In an acute inhalation toxicity study (17/053-004P), a group of young adult CRL: (WI) Wistar strain rats (5/sex) were exposed to a test atmosphere of 50 % w/w TiBTD (97%) formulated in acetone for 4 hours (nose only) at a target concentration of 5 mg/L. Animals were then observed for 14 days.

LC50 male/female = > 5 mg/L

The mean achieved concentration was 4.96 ± 0.32 mg/L (MMAD: 1.51 µm; GSD: 2.00). The inhalable fraction (<4 µm) was 92 %. No deaths occurred during the study. Laboured respiration (slight) was recorded in 1/10 animals on the day of exposure. The animals were symptom free from Day 1. Slight (0.6 %) body weight loss was shown in one male rat between Day 0 and Day 7. During the observation period, normal body weight gain was observed. Upon necropsy, there were no external or internal findings that could be detected.

This acute inhalation toxicity test in rats is acceptable and satisfies the guideline requirement for an OECD 403 study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LC50
5 mg/m³
Quality of whole database:
There is one key study available and it an OECD 403/GLP study. The quality of the database is high.

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Acute oral toxicity

There is one acute oral toxicity study in rats available.

In an acute oral toxicity study (OECD 423/GLP), Crl:WI Wistar female rats (3/dose) were given TiBTD (97%) in 1% methyl cellulose at doses of 2000 mg/kg bw and observed for 14 days. Initially, 3 females were treated at a dose level of 2000 mg/kg bw. As no mortality was observed, a confirmatory group of 3 females was treated at the same dose level. TiBTD did not cause mortality at a dose level of 2000 mg/kg bw in any animal. All animals were symptom-free during the observation period at a dose level of 2000 mg/kg bw. Body weight gains of TiBTD treated animals during the study showed no indication of a test item-related effect. There was no evidence of the macroscopic observations in animals dosed at 2000 mg/kg bw and subjected to the necropsy on Day 14. The LD50 (female) was >2000 mg/kg bw.

Acute inhalational toxicity

There is one acute inhalational toxicity study in rats available.

In an acute inhalation toxicity study (OECD 403/GLP), a group of young adult CRL: (WI) Wistar strain rats (5/sex) were exposed to a test atmosphere of 50 % w/w TiBTD (97%) formulated in acetone for 4 hours (nose only) at a target concentration of 5 mg/L. Animals were then observed for 14 days. The mean achieved concentration was 4.96 ± 0.32 mg/L (MMAD: 1.51 µm; GSD: 2.00). The inhalable fraction (<4 µm) was 92 %. No deaths occurred during the study. Laboured respiration (slight) was recorded in 1/10 animals on the day of exposure. The animals were symptom free from Day 1. Slight (0.6 %) body weight loss was shown in one male rat between Day 0 and Day 7. During the observation period, normal body weight gain was observed. Upon necropsy, there were no external or internal findings that could be detected. The LC50 male/female was > 5 mg/L.

The results from these tests are suitable to use in the human health hazard assessment.

Justification for classification or non-classification

Based on the available information in the dossier, the substance TiBTD (CAS No. 3064-73-1) does not need to be classified for acute toxicity or specific target organ toxicity - single exposure when the criteria outlined in Annex I of 1272/2008/EC are applied.