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Description of key information

ACUTE TOXICITY: ORAL

The LD50 of the test material in the female Wistar strain rat was estimated to be greater than 2000 mg/kg bodyweight.

ACUTE TOXICITY: DERMAL

The LD50 of the test material in male and female Wistar strain rats was found to be greater than 2000 mg/kg bodyweight.

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:
23 October 2012 - 22 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: RccHan:WIST
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: The bodyweight variation did not exceed ± 20 % of the bodyweight of the initially dosed animal.
- Fasting period before study: The animals were fasted overnight immediately before dosing and for approximately three to four hours after dosing.
- Housing: The animals were housed in groups of up to four in suspended solid-floor polypropylene cages furnished with woodflakes.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum access to mains drinking water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): lighting was controlled by a time switch to give twelve hours continuous light (0600 to 1800) and twelve hours darkness.

IN-LIFE DATES: From: 23 October 2012 To: 22 November 2012
Route of administration:
oral: gavage
Vehicle:
other: distilled water
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 200 mg/mL

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg
Doses:
300 and 2000 mg/kg
No. of animals per sex per dose:
1 female animal was dosed at 300 mg/kg
5 female animals were dosed at 2000 mg/kg
Control animals:
no
Details on study design:
SIGHTING STUDY
In the absence of data regarding the toxicity of the test material, 300 mg/kg was chosen as the starting dose.
A single female animal was treated at a dose level of 300 mg/kg (concentration 30 mg/mL, dose volume 10 mL/kg). No toxicity was observed.
In the absence of toxicity at a dose level of 300 mg/kg, an additional female animal was treated at 2000 mg/kg.

MAIN STUDY
No toxicity was observed in the animal treated at 2000 mg/kg, therefore a further four animals were treated, bringing the number treated at this dose level to 5.
All animals were dosed once by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted bodyweight at the time of dosing. Treatment of animals was sequential. Sufficient time was allowed between each dose level to confirm the survival of the previously dosed animals.

- Duration of observation period following administration: 14 days.
- Frequency of observations and weighing: Clinical observations were made 0.5, 1, 2, and 4 hours after dosing and then daily for fourteen days. Morbidity and mortality checks were made twice daily. Individual bodyweights were recorded on Day 0 (the day of dosing) and on Days 7 and 14.
- Necropsy of survivors performed: yes. At the end of the observation period, the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.
Preliminary study:
In the single animal dosed at 300 mg/kg, no mortality was observed and no signs of systemic toxicity were present. The animal showed expected bodyweight gain throughout the observation period. No abnormalities were detected at necropsy.
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality was observed.
Clinical signs:
At 2000 mg/kg, hunched posture was noted during the day of dosing in the animal that was treated initially. No signs of systemic toxicity were noted in the four additionally treated animals.
No effects observed at 300 mg/kg.
Body weight:
All animals showed the expected gains in bodyweight over the observation period.
Gross pathology:
At 2000 mg/kg, dark kidneys were noted at necropsy of the animal treated initially; no abnormalities were noted at necropsy of the remaining four animals.
No effects observed in the necropsy of the rat dosed at 300 mg/kg
Interpretation of results:
other: not classified according to EU criteria
Conclusions:
The LD50 of the test material in the female Wistar strain rat was estimated to be greater than 2000 mg/kg bodyweight and as such requires no classification in accordance with EU criteria.
Executive summary:

The acute oral toxicity of the test material was assessed in the Wistar strain rat in accordance with the standardised guidelines OECD 420 and EU Method B.1 bis.

In the absence of toxicological information, a single fasted female was dosed at 300 mg/kg bw. In the absence of toxicity, a further sighting test was conducted by administering a dose of 2000 mg/kg to one fasted female. Following this, a further group of four fasted females was given a single oral dose of the test material as a suspension in distilled water at a dose level of 2000 mg/kg bodyweight.

No mortality was seen. The animal initially dosed with 2000 mg/kg bw showed hunched posture on the day of dosing; no other signs of systemic toxicity were seen throughout the study. All animals showed expected gains in bodyweight and no abnormalities were noted at necropsy with the exception of dark kidneys noted in the initially dosed animal.

The acute oral median lethal dose (LD50) of the test material in the female Wistar strain rat was estimated to be greater than 2000 mg/kg bodyweight and as such requires no classification in accordance with EU criteria.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The key study was conducted under GLP conditions in accordance with the standardised guidelines OECD 420 and EU Method B.1 bis. It was assigned a reliability score of 1 in accordance with the criteria detailed by Klimisch (1997).

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Data waiving:
study technically not feasible
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with section 2 of REACH Annex XI, the acute inhalation study (required in Annex VIII section 8.5.2) does not need to be conducted as the testing is not technically possible due to the nature of the substance (hygroscopic). Furthermore, exposure via the inhalation route is not anticipated. Therefore, the acute oral and acute dermal studies are deemed more appropriate to address acute toxicity exposure.
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:
31 October 2012 - 21 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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
- Strain: RccHan:WIST.
- Age at study initiation: 8 - 12 weeks.
- Weight at study initiation: At the start of the study the animals weighed at least 200 g. The weight variation did not exceed ± 20 % of the mean weight for each sex, with the exception of on male, which was heavier than all the others.
- Fasting period before study: No.
- Housing: The animals were housed in suspended solid-floor polypropylene cages furnished with woodflakes, individually during the exposure period and in groups of up to four, by sex, for the remainder of the study.
- Diet (e.g. ad libitum): ad libitum access to rodent diet.
- Water (e.g. ad libitum): Free access to mains drinking water.
- Acclimation period: At least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 °C
- Humidity (%): 30 - 70 % (relative)
- Air changes (per hr): At least fifteen changes per hour.
- Photoperiod (hrs dark / hrs light): lighting was controlled by a time switch to give twelve hours continuous light (0600 to 1800) and twelve hours darkness.

IN-LIFE DATES: From: 31 October 2012 To: 21 November 2012
Type of coverage:
semiocclusive
Vehicle:
other: moistened with distilled water
Details on dermal exposure:
TEST SITE
- Area of exposure: The back and flanks clipped free of hair.
- % coverage: Approximately 10 % of the total body surface area.
- Type of wrap if used: A piece of surgical gauze was placed over the treatment area and semi-occluded with a piece of self-adhesive bandage.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The treated skin and surrounding hair was wiped with cotton wool moistened with distilled water.
- Time after start of exposure: After the 24 hour exposure period, following the removal of the semi-occlusive dressing.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg bodyweight.

VEHICLE
- Amount(s) applied: The test material was moistened with distilled water prior to application.
Duration of exposure:
24 hours
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
not required
Details on study design:
- Treatment schedule: In the absence of data suggesting toxicity, one male and one female were initially treated at a dose level of 2000 mg/kg. These animals remained in individual housing following exposure. As no mortalities were noted, a further group of animals (four males and four females) was similarly treated with the test material at a dose level of 2000 mg/kg bodyweight to give a total of five males and five females.
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were observed for deaths or overt signs of toxicity 0.5, 1, 2 and 4 hours after dosing and subsequently once daily. Individual bodyweights were recorded prior to application of the test material on Day 0 and on Days 7 and 14.
- Necropsy of survivors performed: Yes. At the end of the study the animals were killed by cervical dislocation and subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.
- Other examinations performed: After removal of the dressings and subsequently once daily, the test sites were examined for evidence of primary irritation and scored according to the Draize scale:

Erythema and Eschar Formation Value
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to slight eschar formation (injuries in depth) 4

Oedema Formation
No oedema 0
Very slight oedema (barely perceptible) 1
Slight oedema (edges of area well-defined by definite raising) 2
Moderate oedema (raised approximately 1 millimetre) 3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) 4

Any other skin reactions, if present were also recorded.
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
There was no mortality.
Clinical signs:
Dehydration was noted in one male one day after dosing. No other signs of systemic toxicity were noted.
Body weight:
Individual bodyweights and weekly bodyweight changes are given in Table 1.
Animals showed the expected gains in bodyweight over the study period with the exception of one female which showed minimal bodyweight loss during the first week but expected gain in bodyweight during the second week and one female which showed expected gain in bodyweight during the first week but minimal bodyweight loss during the second week.
Gross pathology:
No abnormalities were noted at necropsy.
Other findings:
DERMAL REACTIONS
Off white residual test material was noted at the test site of one male.
Very slight erythema (score of 1) was noted at the test site of one female three to five days after dosing. There were no signs of dermal irritation noted in the remaining animals.

Table 1: Individual Bodyweights and Weekly Bodyweight Changes

Animal Number and Sex

Bodyweight (g) at Day

Bodyweight Change (g) During Week

0

7

14

1

2

1-0 Male

3-0 Male

3-1 Male

3-2 Male

3-3 Male

254

301

223

247

222

266

312

231

261

226

301

321

243

285

241

12

11

8

14

4

35

9

12

24

15

2-0 Female

4-0 Female

4-1 Female

4-2 Female

4-3 Female

241

210

210

203

200

219

209

219

209

209

216

214

228

219

222

5

-1

9

6

9

-3

5

9

10

13

Interpretation of results:
other: not classified according to EU criteria
Conclusions:
The LD50 of the test material in the Wistar strain rat was found to be greater than 2000 mg/kg bodyweight and as such requires no classification in accordance with EU criteria.
Executive summary:

The acute dermal toxicity potential of the test material was assessed in a limit test conducted in the Wistar strain rat in accordance with the standardised guidelines OECD 402 and EU Method B.3.

Initially, two animals (one male and one female) were given a single, 24 hour, semi-occluded dermal application of the test material to intact skin at a dose level of 2000 mg/kg bodyweight. Based on the results of the initial test, a further group of eight animals (four males and four females) was similarly treated. Clinical signs and bodyweight development were monitored during the 14 day observation period. All animals were subjected to gross necropsy.

There were no deaths and no signs of systemic toxicity, though one male animal was dehydrated one day after dosing. Furthermore, the animals showed the expected gains in bodyweight with the exception of one female which showed minimal bodyweight loss during the first week but expected gain in bodyweight during the second week and one female which showed expected gain in bodyweight during the first week but minimal bodyweight loss during the second week.

The only dermal irritation observed was very slight erythema (score of 1) in one female animal three to 5 days after dosing. No abnormalities were noted at necropsy.

The LD50 of the test material in the Wistar strain rat was found to be greater than 2000 mg/kg bodyweight and as such requires no classification in accordance with EU criteria.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The key study was conducted under GLP conditions in accordance with the standardised guidelines OECD 402 and EU Method B.3. It was assigned a reliability score of 1 in accordance with the criteria detailed by Klimisch (1997).

Additional information

Acute Toxicity: Oral

The acute oral toxicity of the test material was assessed in the Wistar strain rat in accordance with the standardised guidelines OECD 420 and EU Method B.1 bis. The study was conducted under GLP conditions and was awarded a reliability score of 1 in accordance with the criteria of Klimisch (1997).

In the absence of toxicological information, a single fasted female was dosed at 300 mg/kg bw. In the absence of toxicity, a further sighting test was conducted by administering a dose of 2000 mg/kg to one fasted female. Following this, a further group of four fasted females was given a single oral dose of the test material as a suspension in distilled water at a dose level of 2000 mg/kg bodyweight.

No mortality was seen. The animal initially dosed with 2000 mg/kg bw showed hunched posture on the day of dosing; no other signs of systemic toxicity were seen throughout the study. All animals showed expected gains in bodyweight and no abnormalities were noted at necropsy with the exception of dark kidneys noted in the initially dosed animal.

The acute oral median lethal dose (LD50) of the test material in the female Wistar strain rat was estimated to be greater than 2000 mg/kg bodyweight and as such requires no classification in accordance with EU criteria.

 

Acute Toxicity: Inhalation

In accordance with section 2 of REACH Annex XI, the acute inhalation study (required in Annex VIII section 8.5.2) does not need to be conducted as the testing is not technically possible due to the nature of the substance.

Furthermore, exposure via the inhalation route is not anticipated. Therefore, the acute oral and acute dermal studies are deemed more appropriate to address acute toxicity exposure.

 

Acute Toxicity: Dermal

The acute dermal toxicity potential of the test material was assessed in a limit test conducted in the Wistar strain rat in accordance with the standardised guidelines OECD 402 and EU Method B.3. The study was conducted under GLP conditions and was awarded a reliability score of 1 in accordance with the criteria of Klimisch (1997).

Initially, two animals (one male and one female) were given a single, 24 hour, semi-occluded dermal application of the test material to intact skin at a dose level of 2000 mg/kg bodyweight. Based on the results of the initial test, a further group of eight animals (four males and four females) was similarly treated. Clinical signs and bodyweight development were monitored during the 14 day observation period. All animals were subjected to gross necropsy.

There were no deaths and no signs of systemic toxicity, though one male animal was dehydrated one day after dosing. Furthermore, the animals showed the expected gains in bodyweight with the exception of one female which showed minimal bodyweight loss during the first week but expected gain in bodyweight during the second week and one female which showed expected gain in bodyweight during the first week but minimal bodyweight loss during the second week. 

The only dermal irritation observed was very slight erythema (score of 1) in one female animal three to 5 days after dosing. No abnormalities were noted at necropsy.

The LD50 of the test material in the Wistar strain rat was found to be greater than 2000 mg/kg bodyweight and as such requires no classification in accordance with EU criteria.

Justification for selection of acute toxicity – oral endpoint

Only one study available. This study was considered reliable enough to address the acute toxicity via the oral route endpoint.

Justification for selection of acute toxicity – dermal endpoint

Only one study available. This study was considered reliable enough to address the acute toxicity via the dermal route endpoint.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the test material does not require classification for acute toxicity.