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Diss Factsheets

Administrative data

Description of key information

In the key (a single dose only) acute oral study (Bayer AG, 1979) in male rats, the LD50 was greater than 4800 mg/kg bw. There were no deaths or clinical signs of toxicity at this dose.  

In the key acute inhalation study (RCC, 1994), rats were exposed to a nose only single 4-hour exposure of a mixture of vapour and liquid D4. All animals showed initial body weight loss and slightly reduced food intakes. Hunched posture, stiff gait, ruffled fur were noted in all groups with restlessness and/or excitement during exposure among those animals that died. At necropsy, decedents had a red discoloration of the lungs which was likely due to the aerosol exposure. The LC50 was calculated to be 36 mg/L air/4h, corresponding to an atmosphere of 2975 ppm.  

The LD50 in the key dermal study (Bayer AG, 1985) in rats was >2.5 ml/kg bw (>2375 mg/kg bw based on density of 0.95 g/cm3). No deaths occurred and no clinical signs were observed following a single application of undiluted D4.  

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:
1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Principles of method if other than guideline:
A single oral administration of 5 ml/kg of test substance to 10 male rats. Observation period 14 days.
GLP compliance:
no
Test type:
other: LD50
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS

- Source: Wikelmann

- Weight at study initiation: 160-180g

- Housing: 5 animals per cage



Route of administration:
oral: unspecified
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
No detail available.

Doses:
5.0 ml/kg bw
No. of animals per sex per dose:
10M
Control animals:
not specified
Details on study design:
- Duration of observation period following administration: 14 days

- Frequency of observations and weighing: no detail available

- Necropsy of survivors performed: not specified

- Other examinations performed: clinical signs, body weight, organ weights, histopathology, other: No detail available.
Statistics:
No statistical analysis reported.
Key result
Sex:
male
Dose descriptor:
LD50
Effect level:
> 4 800 mg/kg bw
Based on:
test mat.
Remarks on result:
other: > 5.0 ml/kg bw (> 4800 mg/kg bw)
Mortality:
No mortality.
Clinical signs:
other: No clinical signs.
Gross pathology:
None reported.
Other findings:
None reported.
Interpretation of results:
GHS criteria not met
Conclusions:
An acute oral LD50 value of > 5 ml/kg (ca. 4800 mg/kg) was determined in male rats in a reliable non-GLP study conducted according to an appropriate test protocol.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
4 800 mg/kg bw

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:
1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
GLP compliance:
yes
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS

- Source: Charles River WIGA GmbH, Sandhoferweg 7, D-97633 Sulzfeld, Germany

- Age at study initiation: 6-8 weeks

- Weight at study initiation: 150-166g males, 103-116g females

- Housing: Animals were housed individually in Makrolon type-3 cages, with standard softwood bedding

- Diet: pelleted standard Kilba 343 rat maintenance diet, ad libitum

- Water: community tap water (Geneva), ad libitum

- Acclimation period: 7-13 days


ENVIRONMENTAL CONDITIONS

- Temperature (°C): 22 +/-3C

- Humidity (%): 30-70

- Air changes (per hr): 10-15

- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: none
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

- Exposure apparatus: Inhalation exposure was performed according to the method of Sachsse et al. (1973, 1976). The test article stream reached the animals noses through ports situated at different levels around the axis of the chamber. Each level had 8 ports and could be rotated, allowing close observation of all the animals without interruption of exposure.

- Exposure chamber volume: The internal active volume of the exposure chamberfor exposing 24 animals is ca. 0.7 litres.

- Method of holding animals in test chamber: The animals were confined separately in Makrolon tubes which were positioned radially around the exposure chamber.

- Source and rate of air: Mean airflow rate was 1.2 l air/minute per animal (group 1), 1.0 l air/minute per animal (group 2) and 0.8 l air/minute per animal (group 3). The time for the concentration to reach 99% of its ultimate value (T99) at an animal port is 7 seconds.

- Method of conditioning air: The flow past, nose only design of this exposure system is based upon the fluid dynamic modelling of the aerosol flow. It ensures a uniform test article distribution, provides a constant stream of 'fresh' test article to each animal, and precludes rebreathing the exhaled air.

- System of generating particulates/aerosols: The test article was placed as supplied in a glass flask feeding a nebulizer. The level of test article in the reservoir of the nebulizer was kept constant and was generated at room temperature. The test atmosphere generated by the nebulizer was diluted with compressed filtered air to achieve the concentrations required for this study and discharged into the exposure chamber with a target mass median aerodynamic diameter of 4 micrometres or less.

- Temperature, humidity, pressure in air chamber: Samples for the measurement of the test article concentration (analytical, gravimetric), oxygen concentration, relative humidity and temperature were collected from a port of the exposure chamber, directly from the feed tube delivering 'resh' test article to the animals nose. Therefore, all the measurements were iso axial and represented exactly what was delivered to the animals.


TEST ATMOSPHERE

- Brief description of analytical method used: The analytical determinations of the test article in the test atmosphere were performed using test atmosphere samples collected in wash-bottles connected to the exhaust of the Gelman filter holder (used for gravimetric determinations). The volatile phase of the test atmosphere was passed into three wash bottles placed in series containing each 80ml of n-hexane cooled at -70C. The content of each wash bottle was transferred into 100ml volumetric flasks, the wash bottles were rinsed with hexane and the flasks were made up to 100ml with the rinsing. Aliquots of each wash bottle were put into appropriate sealed vials and sent for analysis at ambient temperature.

- Samples taken from breathing zone: yes




TEST ATMOSPHERE (if not tabulated)

- MMAD (Mass median aerodynamic diameter): 4 micrometres or less.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
20.12 mg/l; 30.03 mg/l; 54.37 mg/l
No. of animals per sex per dose:
5/sex/dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days

- Frequency of observations and weighing: Clinical signs and mortality were noted during and following the exposures over a 15-day observation period. Body weights were recorded once during the acclimatisation period and then on test days 1 (prior to exposure), 2, 3, 4, 5, 9, 12 and 15. Food consumption was measured in the rats which survived the treatment once during acclimatisation period (over 5 days), then during 4 intervals following treatment (from days 1-6, 6-9, 9-12 and 12-15).

- Necropsy of survivors performed: yes

- Other examinations performed: clinical signs, body weight, organ weights, histopathology, other: All macroscopical abnormalities following necropsy were recorded. The lungs, liver, spleen and thymus of all surviving animals were weighed before fixation.
Statistics:
The LOGIT-Model (LOGIT: A program for Dose-response Analysis, koshiver J. and Moore D., Computer Programs in Biomedicine, 10, 1979, 61-75) was used to calculate LC50.
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
36 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: (corresponding to approx. 2,975 ppm)
Mortality:
See Table 3.
Clinical signs:
other: Reduced food consumption in all groups. Hunched posture, stiff gait, ruffled fur in all groups; restlessness and/or excitement during exposure in all animals that died; tachypnea (mid dose), rales and/or head drop (some low and mid dose males). Following
Body weight:
All animals of the low and medium exposure groups as well as the surviving male of the high exposure group lost weight during the day of exposure (in a few cases during two to three days) following exposure. This reduction in body weight was slightly more pronounced in the medium than in the low exposure group. After 9 days the body weights of the surviving animals were again close to pre-exposure values. The growth rate until the end of the 15 day observation period was considered to remain slightly under normal values.
Gross pathology:
No macroscopic abnormalities were noted in any animals of the low exposure group. The lungs of all decedent animals of the medium and high exposure groups showed red discoloration. In addition, dark red or reddish foci were seen on the thymus of 1 male and 1 female of the medium exposure group, and of one male of the high exposure group. The mandibular lymph nodes of 1 high exposure male showed a reddish discolouration. Changes in the lungs are related to spontaneous death due to the treatment. Changes in the thymus and in the lymph nodes are considered to be incidental.
Other findings:
- Organ weights: All organ weight differences (if any) between groups were marginal. Trend of lung weight increase in both sexes, thymus weight decrease in mass, and spleen weight increase in both sexes. Data from a single animal were not considered as sufficient for accurate evaluation of organ weight changes at the high exposure level.
Interpretation of results:
GHS criteria not met
Conclusions:
An acute inhalation LC50 value of 36 mg/l/4h air was determined in both male and female rats in a reliable study conducted according to an appropriate protocol, and in compliance with GLP.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LC50
Value:
36 000 mg/m³ air

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1984/08/15-1984/08/29
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
GLP compliance:
no
Test type:
other: LD50
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS

- Source: Winkelmann, Borchen

- Age at study initiation: 9-14 weeks

- Weight at study initiation: ca. 180g

- Housing: Conventional Makrolon type 3 cages

- Diet: Altromin R 1324 ad libitum

- Water: ad libitum




ENVIRONMENTAL CONDITIONS

- Temperature (°C): 22 +/- 1.5

- Humidity (%): 60 +/-5

- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:
not specified
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST MATERIAL

- Amount(s) applied (volume or weight with unit): 2.5ml/kg bw


Duration of exposure:
Single instillation
Doses:
no data
No. of animals per sex per dose:
5 per sex
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days

- Frequency of observations and weighing: The animals were observed twice daily during the 14 day observation period, and the beginning and intensity of symptoms recorded. The animals were weighed in the beginning, middle and end of the observation period.

- Necropsy of survivors performed: yes

- Other examinations performed: clinical signs, body weight, organ weights, histopathology, other: all animals were subjected to gross necropsy.
Statistics:
No statistical analysis was carried out.
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2.5 mL/kg bw
Based on:
test mat.
Remarks on result:
other: (>2375 mg/kg) bw
Mortality:
No mortality.
Clinical signs:
other: No clinical signs.
Gross pathology:
No treatment-related findings at necropsy.
Other findings:
None reported.
Interpretation of results:
GHS criteria not met
Conclusions:
An acute dermal LD50 value of >2.5 ml/kg (ca. 2375 mg/kg bw) was determined in a reliable study conducted according to an appropriate test protocol. Not conducted according to GLP.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 375 mg/kg bw

Additional information

In the key oral study (Bayer AG, 1979), Wistar rats (10 male) were administered with a single dose of 5.0 ml/kg bw D4 (4800 mg/kg bw). The animals were then observed for 14 days for mortality and signs of toxicity. At the end of the observation period the animals were sacrificed and a histopathological examination conducted and organ weights recorded (organs not specified). There were no adverse findings reported. Other results from supporting studies in rats are in agreement with the conclusion that D4 is not harmful via the oral route. A Reliability 2 study in mice (Pasquet and Mauret, 1971) gave an LD50 of 1700 mg/kg bw. However, this value is not selected as the key study since rat is the preferred species for classification and labelling purposes.

In the key inhalation study (RCC, 1994) Fischer 344 rats (5 animals/sex/concentration) were exposed to D4 aerosol (nose-only) at concentrations of 20.12 mg/l, 30.03 mg/l and 54.37 mg/l for 4 hours. The animals were then observed for 14 days for mortality and signs of toxicity. Body weight and food consumption were recorded. At the end of the observation period the animals were sacrificed, and macro- and microscopic examinations conducted. The lungs, liver, spleen and thymus of all surviving animals were weighed before fixation. Mortality occurred in mid and high dose groups. Reduced food consumption was observed in all groups. Clinical signs that were seen in all groups included hunched posture, stiff gait and ruffled fur. The animals that survived appeared normal after 6 days following exposure. All surviving animals had reduced body weight on the day of exposure which was considered to remain slightly under normal values by the end of the observation period. There were no adverse findings observed at necropsy. The lungs of all decedent animals of the medium and high exposure groups showed red discoloration which were related to spontaneous death due to the treatment.

The LC50 was calculated to be 36 mg/L air/4hour, which is above the concentration cut-off limits for classification for acute inhalation toxicity, therefore D4 is not considered to be acutely toxic via inhalation route.

A number of other studies, including non-standard exposures to saturated vapour, support the conclusion that D4 is not acutely harmful via the inhalation route.

In the key dermal study (Bayer, 1985) 2.5ml/kg (2375 mg/kg bw) of undiluted D4 was applied to the skin of Wistar rats (5 animals/sex). There was no information on how long D4 was applied for, or what type of dressing (if any) was used. The animals were then observed for 14 days for mortality and clinical signs of toxicity. The animals were also weighed during the observation period. At the end of the observation period the animals were examined macro- and microscopically. There were no adverse findings reported. A number of supporting studies (Reliability 2 or 4) confirm that D4 is not acutely harmful via the dermal route.


Justification for classification or non-classification

Based on the available data for oral, inhalation and dermal routes, D4 does not require classification for acute toxicity according to Regulation (EC) No. 1272/2008.