Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates

Endpoint summary

Description of key information

Oral (OECD 423), rat (female): LD50 > 300 < 2000 mg/kg bw; LD50 (cut-off) = 500 mg/kg bw

Dermal (OECD 402), rat (male/female): LD50 > 2000 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:

20 Aug - 19 Sep 2013

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:

8 Feb 2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.1000 (Acute toxicity testing background)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 8 - 11 weeks
- Weight at study initiation: 144 - 154 g (animals 1-3; step 1), 170 - 178 g (animals 4-6; step 2), 150 - 165 g (animals 7-9; step 3) and 152 - 160 g (animals 10-12; step 3)
- Fasting period before study: 16 to 19 h (access to water was permitted)
- Housing: animals were kept in groups in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 030512).
- Diet: Altromin 1324 maintenance diet for rats and mice (lot no. 0939), ad libitum
- Water: tap water, sulphur acidified to a pH value of approximately 2.8, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: aqua ad injectionem (sterile water)

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 0.2 g/mL (2000 mg/kg bw dose groups) and 0.03 g/mL (300 mg/kg bw dose groups)
- Amount of vehicle: 10 mL/kg bw
- Justification for choice of vehicle: the vehicle was chosen due to its non-toxic characteristics.
- Lot/batch no.: 10952-1

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

DOSAGE PREPARATION: For all animals of step 1 and step 2, 1.5 g of the test item were emulsified with the vehicle to gain a final volume of 7.5 mL and to achieve a dose of 2000 mg/kg body weight at a dose volume of 10 mL/kg body weight. For all animals of step 3 and step 4, 0.3 g of the test item were emulsified with the vehicle to gain a final volume of 10 mL and to achieve a dose of 300 mg/kg body weight at a dose volume of 10 mL/kg body weight. The dose formulations were made shortly before each dosing occasion. Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension for 10 min before every dose administration.

Doses:

Step 1 and 2: 2000 mg/kg bw
Step 3 and 4: 300 mg/kg bw

No. of animals per sex per dose:

3 per step

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: animals were weighed on Day 1 (prior to the administration) and on Days 8 and 15. A careful clinical examination was made several times on the day of dosing (at least once during the first 30 minutes and with special attention given during the first 4 hours post-dose). Thereafter, animals were observed for clinical signs once daily until the end of the 14-day observation period.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 300 - < 2 000 mg/kg bw

Based on:

test mat.

Sex:

female

Dose descriptor:

LD50 cut-off

Effect level:

500 mg/kg bw

Based on:

test mat.

Mortality:

Step 1 (2000 mg/kg bw): 1/3 females died
Step 2 (2000 mg/kg bw): 3/3 females died
Step 3 and 4 (300 mg/kg bw): 0/3 females died

Clinical signs:

other: 2000 mg/kg bw: reduced spontaneous activity, moving the bedding, kyphosis, bradykinesia, piloerection, tremor and salivation; in the surviving animals of this dose group, all symptoms recovered within up to 2 days post dose. 300 mg/kg bw: reduced spontane

Gross pathology:

Necropsy revealed no substance-related findings in surviving animals. Macroscopic findings of the four animals found dead at 2000 mg/kg bw involved bleeding nose (1/4 dead animals) and a bloated stomach (4/4 dead animals) containing remains of the test item (3/4 dead animals).

Table 1. Clinical signs and mortality - acute oral toxicity

Step	Dose [mg/kg bw]	Toxicological results*	Duration of clinical signs	Time of death (dead animals/3 animals)	Mortality (%)
1	2000	1/3/3	5 min - Day 2	2 h 10 min	33
2	2000	3/3/3	10 min - death	1 h 40 min (1/3) + Day 2 (2/3)	100
3	300	0/3/3	5 min - Day 2	---	0
4	300	0/3/3	10 - 30 min	---	0
LD50 cut-off = 500 mg/kg bw

  * first number = number of dead animals, second number = number of animals with clinical signs, third number = number of animals used

         

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

CLP: Acute Oral 4, H302

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

500 mg/kg bw

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:

25 Jul - 16 Aug 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

adopted Feb 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 240 - 277 g (males); 215 - 231 g (females)
- Housing: The initial two animals were housed individually throughout the study. The further group of eight animals (four male and four female) were housed individually during the 24-hour exposure period and in groups of four, by sex, for the remainder of the study.
- Diet: 2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK, ad libitum
- Water: drinking water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 Jul 2017 To: 16 Aug 2017

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: 6 x 6 cm² clipped skin of the dorsal area of the trunk
- % coverage: 10
- Type of wrap if used: the test material was held in contact with the skin with surgical gauze and a piece of self adhesive bandage

REMOVAL OF TEST SUBSTANCE
- Washing: residual test material was removed with cotton wool moistened with arachis oil BP
- Time after start of exposure: 24 h

Duration of exposure:

24 h

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were observed for deaths or overt signs of toxicity 30 min, 1, 2 and 4 h after dosing and subsequently once daily for 14 days. Body weights were recorded prior to treatment and on Day 7 and 14 after dosing.
- Necropsy of survivors performed: Yes, all animals were subjected to gross necropsy.
- Other examinations performed: After removal of the dressings and subsequently once daily for 14 days, the test sites were examined for evidence of primary irritation and scored according to Draize scoring system.

Preliminary study:

One male and one female rat were initially treated with the test item at a dose level of 2000 mg/kg bw. As no mortalities were noted a further group of animals (four males and four females) was similarly treated with the test item at a dose level of 2000 mg/kg bw to give a total of five males and five females.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occurred during the study period.

Clinical signs:

other: No clinical signs of toxicity were observed up to the end of the 14-day observation period.

Gross pathology:

No abnormalities were noted at necropsy.

Other findings:

- Primary irritation: Signs of dermal irritation included very slight to well defined erythema, slight edema, scattered areas of green necrosis, scattered areas of blanching, small scattered superficial scabs, hardened light and dark brown colored scabs, scab cracking, scab lifting to reveal glossy skin, dried blood and/or bleeding, crust formation and desquamation. Adverse dermal reactions prevented the accurate evaluation of erythema and edema at the treatment sites of two male and two female animals.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

Data on acute oral toxicity and on acute dermal toxicity are available for Amines C12-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 96690-34-5) in Wistar rats to fulfill the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VII-VIII, 8.5.

Oral

The acute oral toxicity of Amines, C12-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 96690-34-5) was studied in female Crl: WI(Han) Wistar rats according to the acute toxic class method (OECD guideline 423) and under the requirements of GLP (BSL Bioservice, 2012). In a first step, 3 animals each received formulations of test substance in aqua ad injectionem (sterile water) at 2000 mg/kg bw/day via oral gavage. Since 1/3 animals died during the first step, application of the test substance at 2000 mg/kg bw to 3 further animals was repeated in a second step, resulting in mortality of all treated animals. In a subsequent third and fourth step, 3 additional animals per step received the test substance at a lower dose level of 300 mg/kg bw, which resulted in no mortalities in all treated animals of both treatment steps. The most relevant clinical signs in animals treated at 2000 mg/kg bw involved reduced spontaneous activity, moving the bedding, kyphosis, bradykinesia, piloerection, tremor and salivation. Animals treated at a dose level of 300 mg/kg bw showed reduced spontaneous activity, moving the bedding, kyphosis, piloerection, half eyelid-closure and salivation. All clinical findings were fully reversible up to 2 days post application in all surviving animals of both dose groups. Throughout the 14-day observation period, the weight gain of the surviving animals was within the normal range of variation for this strain. At necropsy, no treatment-related macroscopic findings were observed in any of the surviving animals. Macroscopic findings of the four animals found dead at 2000 mg/kg bw in the first two steps involved bleeding nose (1/4 dead animals) and a bloated stomach (4/4 dead animals) containing remains of the test item (3/4 dead animals). Based on the results of the study, the oral LD50 value for female Crl: WI(Han) Wistar rats is > 300 - < 2000 mg/kg bw. In accordance with OECD guideline 423, the oral LD50 cut-off of the test substance is considered to be 500 mg/kg bw and the substance is classified as harmful if swallowed.

 

Dermal

An acute dermal toxicity study (limit test) was performed with the test substance according to OECD guideline 402 and GLP conditions (Envigo Research Limited, 2017). 5 male and 5 female Wistar rats were exposed to 2000 mg test substance /kg bw for 24 h on the back skin under semi-occlusive conditions. The observation period was 14 days. No mortality was observed and there were no systemic clinical signs noted in any animal throughout the study. Body weight gains and necropsy at study termination revealed no abnormalities. Signs of dermal irritation noted included very slight to well defined erythema, slight edema, scattered areas of green necrosis, scattered areas of blanching, small scattered superficial scabs, hardened light and dark brown colored scabs, scab cracking, scab lifting to reveal glossy skin, dried blood and/or bleeding, crust formation and desquamation. Adverse dermal reactions prevented the accurate evaluation of erythema and edema at the treatment sites of two male and two female animals. Thus, the acute dermal LD50 in rats was found to be greater than 2000 mg/kg bw.

Justification for classification or non-classification

The available data on acute toxicity of the substance via the oral route meet the criteria for classification as Acute Oral 4 (H302) according to Regulation (EC) 1272/2008.

The available data on acute toxicity of the substance via the dermal route do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.