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Description of key information

In a reliable acute oral toxicity study performed according to OECD 423 and according to GLP principles, the LD50 cut-off value was determined to be 500 mg/kg bw and in a reliable acute dermal toxicity study performed similar to OECD 402 and according to GLP principles, the LD50 was determined to be >500 and <1000 mg/kg bw.

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:
September - December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Version / remarks:
2008, inlcuding most recent amendments
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Version / remarks:
2002
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000, including the most recent partial revisions.
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
no
Species:
rat
Strain:
other: Crl:WI (Han)
Sex:
female
Details on test animals or test system and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 10-11 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (158 - 201 grams).
- Fasting period before study: Animals were deprived of food overnight prior to dosing and until 3-4 hours after administration of the test item.
- Housing: Group housing of 3 animals per cage in labeled Macrolon cages containing sterilized sawdust as bedding material and paper as cage-enrichment.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Set to maintain:
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): approx 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 September 2015 to 22 October 2015
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
specific gravity 1.036
Details on oral exposure:
GAVAGE METHOD: plastic feeding tubes.

Frequency: single dosage, on Day 1.

VEHICLE
- Justification for choice of vehicle: The vehicle was selected based on trial formulations performed at WIL Research Europe and on test substance data supplied by the sponsor.
- Stability for at least 6 hours at room temperature; 8 days in the refrigerator and 3 weeks in the freezer is confirmed over the concentration range 1 to 200 mg/mL

DOSAGE PREPARATION
The preparations (w/w) were dosed within 4 hours after adding the vehicle to the test item. Homogeneity was assessed by visual inspection of the solutions and the formulations were stirred during dosing, which ensures homogeneity sufficient for these kinds of studies. Adjustment was made for specific gravity of the vehicle. No correction was made for purity of the test item. The concentration of the test item in vehicle was varied to allow constant dosage volume in terms of mL/kg body weight.

The toxicity of the test item was assessed by stepwise treatment of groups of 3 females. The first group was treated at a dose level of 300 mg/kg bw. The absence or presence of mortality of animals dosed at one step determined the next step, based on the test procedure defined in the guidelines. The onset, duration and severity of the signs of toxicity were taken into account for determination of the time interval between the dose groups.
Doses:
300 mg/kg (10 mL/kg) bw
2000 mg/kg (10 mL/kg) bw

No. of animals per sex per dose:
300 mg/kg bw: 6 (2 groups of three females in a stepwise manner)
2000 mg/kg bw: 3
Control animals:
no
Details on study design:
Duration of observation period following administration: 14 days

Frequency of observations and weighing:
- Mortality/Viability: Twice daily. The time of death was recorded as precisely as possible.
- Body weights: Days 1 (pre-administration), 8 and 15.
- Clinical signs: At periodic intervals on the day of dosing (Day 1) and once daily thereafter, until Day 15.

Necropsy of survivors performed: The animals surviving to the end of the observation period were sacrificed by oxygen/carbon dioxide procedure. All animals assigned to the study were subjected to necropsy and descriptions of all internal macroscopic abnormalities recorded.

Other examinations performed: none.
Statistics:
No statistical analysis was performed (The method used is not intended to allow the calculation of a precise LD50 value).
Key result
Sex:
female
Dose descriptor:
LD50 cut-off
Effect level:
500 mg/kg bw
Based on:
test mat.
Mortality:
At 2000 mg/kg bw, all animals were found dead on day 1.
At 300 mg/kg bw, no mortality occurred.
Clinical signs:
At 2000 mg/kg bw, hunched posture and salivation were noted for all animals on Day 1.
At 300 mg/kg bw, lethargy, hunched posture, uncoordinated movements, piloerection and/or ptosis were noted for the animals between Days 1 and 3.
Body weight:
The mean body weight gain shown by the surviving animals over the study period was considered to be similar to that expected for normal untreated animals of the same age and strain.
Gross pathology:
No abnormalities were found at macroscopic post mortem examination in any of the animals.
Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
In an acute oral toxicity study with rats, performed according to OECD/EC test guidelines, an oral LD50 value of Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt was established to be within the range of 300-2000 mg/kg bw. According to the OECD 423 test guideline, the LD50 cut-off value was considered to be 500 mg/kg bw. According to GHS criteria (2011) (including all amendments), Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt should be classified as: harmful if swallowed (Category 4) for acute toxicity by the oral route.
Executive summary:

An assessment of acute oral toxicity with Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt in the rat (Acute Toxic Class Method) was performed according to OECD guidelines and in accordance with GLP principles. Initially, Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt was administered by oral gavage to three female Wistar rats at 300 mg/kg bw. In a stepwise procedure two additional groups of three females were dosed at 2000 and 300 mg/kg bw. At 2000 mg/kg, all animals were found dead on day 1. At 300 mg/kg bw, no mortality occurred. At 2000 mg/kg bw, hunched posture and salivation were noted for all animals on Day 1. At 300 mg/kg bw, lethargy, hunched posture, uncoordinated movements, piloerection and/or ptosis were noted for the animals between days 1 and 3. The mean body weight gain shown by the surviving animals over the study period was considered to be similar to that expected for normal untreated animals of the same age and strain. No abnormalities were found at macroscopic post mortem examination in any of the animals.

The oral LD50 value of Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt in Wistar rats was established to be within the range of 300-2000 mg/kg bw.

According to the OECD 423 test guideline, the LD50 cut-off value was considered to be 500 mg/kg bw.

Based on these results:

- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments), Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt should be classified as: harmful if swallowed (Category 4) for acute toxicity by the oral route;

- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt should be classified as Category 4 and should be labeled as H302: Harmful if swallowed.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
500 mg/kg bw
Quality of whole database:
Study was performed according to OECD 423 guideline (2001) and in accordance with GLP principles.

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

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:
14 June 2006 - 18 June 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study performed according to GLP principles and similar to OECD 402. Minor deviations include lack of gross pathology, animals were only observed for 7 days instead of recommended 14 days.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
yes
Remarks:
no gross pathology, observation time only 7 days (instead of 14 days), acclimatization period 3 days only (instead of recommended 5); test substance covered occlusively (semi-occlusive is recommended).
GLP compliance:
yes
Test type:
standard acute method
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Millbrook Breeding labs, Amherst, MA, USA
- Age at study initiation: 2-3 months old
- Weight at study initiation: 2.1-2.8 kg
- Fasting period before study: No
- Housing: individually in suspended wire cages
- Diet: PMI Rabbit Chow, provided daily
- Water: ad libitum
- Acclimation period: at least three days

ENVIRONMENTAL CONDITIONS
- No further data, but animals kept in climate controlled room
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 June 2006 To: 18 July 2006
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
- % coverage: appr. 10%
- Type of wrap if used: four layered surgical gauze patch, wrapped with plastic sheeting secured with non-irritating tape.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no (test substance was wiped of the skin)
- Time after start of exposure: 24 hours

TEST MATERIAL
- Amount applied: 2.4-2.7 g/ animal, 0.63-0.70 g/ animal, or 1.05-1.3 g/ animal
- For solids, paste formed: no, but patches were moistened with 6-13 ml of distilled water
Duration of exposure:
24 hours
Doses:
1000, 250 or 500 mg/kg bw
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 7 days
- Frequency of observations and weighing: the animals were observed at 1, 4 and 24 hours postdose for mortality, toxicity and pharmacological effects, and once daily thereafter for mortality. Body weights were recorded pretest and at death.
- Necropsy of survivors performed: no
- Other examinations performed: test sites were scored for dermal irritation at 24 hours post-dose and on day 7.
Statistics:
None.
Sex:
male
Dose descriptor:
LD50
Effect level:
> 500 - < 1 000 mg/kg bw
Based on:
test mat.
Mortality:
No animals survived the 1000 mg/ kg bw dose (4/5 animals died between 1 and 4 hours postdose, one animal died between 4 and 24 hours postdose). Four rabbits survived at 500 mg/ kg bw, one rabbit died between 4 and 6 hours post-dose. No mortality occurred at 250 mg/ kg bw.
Clinical signs:
At 250 mg/ kg bw no clinical signs were noted, except for instances of soiling of the anogenital area, ataxia and hunched posture 24 post dose. At 500 mg/ kg bw, tachypnea was observed in one animal one hour after start of exposure. One animal was noted to be lethargic, to have an abnormal posture an decreased responses in its hind legs at unwrapping. At the highest dose hunched posture was seen in the animal that died betwen 4 and 24 hours after exposure.
Body weight:
One animal dosed at 500 mg/kg bw/ day lost weight between day 1 and day 7 (appr. -7.7%), one animal remained at the same body weight. The body weight gain in the other surviving animals increased as expected.
Gross pathology:
Not performed.
Other findings:
At 24 hours, no erythema (in 1/4 animals), very slight (in 2/4 animals) to well defined (in 1/4 animals) was seen at 500 mg/kg bw. No edema was seen in 1/4 animals, two had very slight edema and one had slight edema (edges of area well defined by definitive raising). At day 7, 3/4 had flaking skin with very slight (1/4), well defined (1/4) or severe erythema (beet redness). Edema was absent (2/4), very slight or slight (1/4 each).
Exposure to 250 mg/kg bw resulted at 24 hours in very slight (in 3/5 animals) to well defined (in 2/5 animals) erythema. Edema was absent in 1 animal, very slight (2/5) or slight (2/5). On day 7, only 1/5 rabbits had very slight edema, 4/5 had no erythema (one of these animals had flaking skin) and one animal was found to have severe erythema.

After occlusive binding was removed, approximately 10% of the test substance (both concentrations) remained on the skin (estimated by visual inspection).

Interpretation of results:
other: classified cat. 3
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Based on an acute dermal toxicity test performed with male rabbits, the LD50 of the test substance was found to be > 500 mg/kg bw and < 1000 mg/kg bw.
Executive summary:

An acute dermal toxicity test with male rabbits was performed under GLP principles. Male rabbits were exposed occlusively for 24 hours to 250, 500 or 1000 mg/kg bw and observed for 7 days after removal of the test substance. At the highest dose, all rabbits died within 24 hours. At 500 mg/ kg bw, 1/5 rabbits died. No other mortality occurred. Clinical signs were limited, incidental findings of ataxia and soiling of the anogenital area were seen (at 250 mg/ kg bw) and tachypnea (in the rabbit that died at 500 mg/kg bw) and lethargy, abnormal posture and responsiveness in the hind legs (1 animal) were seen at 500 mg/kg bw. Body weight reduction and arrest were seen in 2 animals at 500 mg/kg bw, all other surviving animals gained weight as expected. Dermal effects were seen in most animals, for one animal from each dose group the erythema was severe at day 7. For all other animals, skin effects were absent or very slight after 7 days.

Based on these results, the LD50 of the test substance was found to be > 500 mg/kg bw and < 1000 mg/ kg bw and therefore the test substance is classified for acute dermal toxicity category 3.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
500 mg/kg bw
Quality of whole database:
Study performed according to GLP principles and similar to OECD 402. Minor deviations include lack of gross pathology, animals were only observed for 7 days instead of recommended 14 days.

Additional information

Acute oral toxicity:

In the available reliable study (Klimisch 1), an assessment of acute oral toxicity with Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt in the rat (Acute Toxic Class Method) was performed according to OECD guidelines and in accordance with GLP principles. Initially, Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt was administered by oral gavage to three female Wistar rats at 300 mg/kg bw. In a stepwise procedure two additional groups of three females were dosed at 2000 and 300 mg/kg bw. At 2000 mg/kg, all animals were found dead on day 1. At 300 mg/kg bw, no mortality occurred. At 2000 mg/kg bw, hunched posture and salivation were noted for all animals on Day 1. At 300 mg/kg bw, lethargy, hunched posture, uncoordinated movements, piloerection and/or ptosis were noted for the animals between days 1 and 3. The mean body weight gain shown by the surviving animals over the study period was considered to be similar to that expected for normal untreated animals of the same age and strain. No abnormalities were found at macroscopic post mortem examination in any of the animals. The oral LD50 value of Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt in Wistar rats was established to be within the range of 300-2000 mg/kg bw. According to the OECD 423 test guideline, the LD50 cut-off value was considered to be 500 mg/kg bw.

 

Acute dermal toxicity:

In the available reliable GLP-compliant study (Klimisch 2), male rabbits were exposed occlusively for 24 hours to 250, 500 or 1000 mg/kg bw and observed for 7 days after removal of the test substance. At the highest dose, all rabbits died within 24 hours. At 500 mg/kg bw, 1/5 rabbits died. No other mortality occurred. Clinical signs were limited, incidental findings of ataxia and soiling of the anogenital area were seen (at 250 mg/ kg bw) and tachypnea (in the rabbit that died at 500 mg/kg bw) and lethargy, abnormal posture and responsiveness in the hind legs (1 animal) were seen at 500 mg/kg bw. Body weight reduction and arrest were seen in 2 animals at 500 mg/kg bw, all other surviving animals gained weight as expected. Dermal effects were seen in most animals, for one animal from each dose group the erythema was severe at day 7. For all other animals, skin effects were absent or very slight after 7 days. Based on these results, the LD50 of the test substance was found to be > 500 mg/kg bw and < 1000 mg/ kg bw.

Justification for classification or non-classification

According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments), Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt should be classified as: harmful if swallowed (Category 4) for acute toxicity by the oral route and toxic in contact with skin (Category 3) for acute toxicity by the dermal route.

According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt should be classified as Category 4 and should be labeled as H302: Harmful if swallowed for oral route and classified as Category 3 and labeled as H311: Toxic in contact with skin for the dermal route