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Description of key information

Two in vivo studies are available performed according to OECD 423 guideline and GLP principles. 

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:
06 May, 2014 - 18 June, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
(2001)
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
no
Species:
mouse
Strain:
other: Crl:CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: Young adult animals (approx. 9 - 12 weeks old)
- Weight at study initiation: 28.8 to 35.2 g for males and 21.9 to 29.9 g for females
- Fasting period before study: 3 to 4-hour period prior to dosing and the approximate 4-hour period after dosing when food was withheld.
- Housing: All animals were housed individually in clean, solid bottom cages containing ground corncob nesting material.
- Diet: Free access to basal diet (PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002)
- Water: Free access to municipal water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7 – 21.4
- Humidity (%): 40.2 - 50.1
- Air changes (per hr): minimum 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
GAVAGE METHOD: plastic feeding tubes.

Frequency: single dosage, on Day 1.

MAXIMUM DOSE VOLUME APPLIED: 1.19 mL/kg. The dose volume was determined by dividing each dose level, expressed as g/kg, by the density (1.6813 g/mL) (dose volumes of 0.0297, 0.178 and 1.19 mL/kg bw for the 50, 300 and 2000 mg/kg bw groups, respectively).

DOSAGE PREPARATION: Prior to use, the bulk test substance container was inverted and/or swirled. On each day of dosing, a sufficient volume of test substance was transferred into an amber glass vial. A stir bar was added, and the contents stirred throughout use. The container was purged with nitrogen and capped immediately. The pH was measured using litmus paper for the first dispensation and was found to be 5.

CLASS METHOD
- Rationale for the selection of the starting dose:
The test substance was administered once to 3 fasted male and 3 fasted female albino mice at a dose level of 2000 mg/kg bw. Mortality was observed for 1 male and 1 female, and 3 additional males and 3 additional females were subsequently dosed at 2000 mg/kg bw. All 3 additional males and all 3 additional females at 2000 mg/kg bw died or were euthanized in extremis. Therefore, 3 males and 3 females were dosed at 300 mg/kg bw. Only 1 male at 300 mg/kg bw was euthanized in extremis, and as called for in the guidelines, 3 additional males were dosed at 300 mg/kg bw. No more unscheduled deaths occurred in the 300 mg/kg bw group males, and therefore, no additional males were dosed. All 3 females dosed at 300 mg/kg bw died or were euthanized in extremis. Therefore, 3 females were dosed at 50 mg/kg bw. No mortality was observed for these females, and 3 additional females were dosed at 50 mg/kg bw. Again, no mortality was observed, and therefore, no additional females were dosed.
Doses:
50, 300 and 2000 mg/kg body weight


No. of animals per sex per dose:
2000 mg/kg bw: 12 (2 groups of three males and three females in a stepwise manner)
300 mg/kg bw: 9 (3 females and 6 males in a stepwise manner)
50 mg/kg bw: 6 (2 groups of three females in a stepwise manner)
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Mortality: The mice were observed at approximately 15 minutes (± 5 minutes) and 1, 2 and 4 hours post-dosing on study day 0 and twice daily, once in the morning and once in the afternoon, thereafter for 14 days.
Clinical observations: The mice were observed at approximately 15 minutes (± 5 minutes) and 1, 2, and 4 hours post-dosing on study day 0 and once daily thereafter for 14 days.
Body weights: Body weights were obtained and recorded on study days 0 (initiation), 7, and 14 (termination). In addition, animals that died on study or needed to euthanized in extremis were weighed as soon as they were found in that condition.
- Necropsy of survivors performed: The moribund (in extremis) mice and all surviving mice on study day 14 were euthanized by carbon dioxide inhalation. The major organ systems of the cranial, thoracic, and abdominal cavities and the eyes and skin were examined for all animals found dead or euthanized.
- 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).
Sex:
male
Dose descriptor:
LD50
Effect level:
> 300 - < 2 000 mg/kg bw
Based on:
test mat.
Sex:
female
Dose descriptor:
LD50
Effect level:
> 50 - < 300 mg/kg bw
Based on:
test mat.
Mortality:
Four of 6 males and 4/6 females at 2000 mg/kg bw and 1/6 males and 3/3 females at 300 mg/kg bw were either found dead or euthanized in extremis. All early deaths were observed between study days 2 through 8. All other animals survived to the scheduled necropsy (study day 14).
Clinical signs:
Clinical observations limited to the early death animals in the 2000 mg/kg bw group consisted of head tilt, tremors, and wet red and clear material around the mouth (post mortem). Cool body or extremities and ataxia were limited to the 300 and 2000 mg/kg bw group early death animals. Dermal atonia was limited to the 300 mg/kg bw early death animals. Other clinical observations noted at 300 and 2000 mg/kg bw, including in animals that survived to study termination, consisted of hypoactivity, decreased defecation, poor body condition (water bottle added), and partial closure of the eye(s). For the animals that survived to the scheduled necropsy, a blue abdomen was observed in the 300 mg/kg bw group males and body weight loss (water bottle added) was noted in the 50, 300, and 2000 mg/kg bw groups. Most of the aforementioned clinical observations occurred between study days 1 and 8.
Body weight:
Three of the 5 surviving males in the 300 mg/kg bw group and both surviving males in the 2000 mg/kg bw group lost weight during the first week after dosing (study days 0 to 7) but then gained weight during the second week after dosing (study days 7 to 14). All surviving males surpassed their study day 0 body weight at the end of the 14-day observation period with the exception of 2000 mg/kg bw group one male. The 6 surviving females in the 50 mg/kg bw group lost a small amount of weight during the first or second week after dosing. Both surviving females in the 2000 mg/kg bw group lost weight during the first week after dosing but then gained weight during the second week after dosing (study days 7 to 14). Three of the surviving females in the 50 mg/kg bw group and both surviving females in the 2000 mg/kg bw group surpassed their study day 0 body weight at the end of the 14-day observation period.
Gross pathology:
Macroscopic findings for early death animals included a small thymus for 3 males and 2 females at 2000 mg/kg bw, a small spleen for 2 males and 2 females at 2000 mg/kg bw, and dark red discoloration of the lungs for 2 females at 300 mg/kg bw and 1 male at 2000 mg/kg bw. In addition, 1 female that was euthanized in extremis at 300 mg/kg bw had dark red areas in the glandular portion of the stomach and dark red contents in the duodenum and jejunum. One female at 300 mg/kg bw that died had dark red contents in the stomach. There were no gross findings for the animals that survived to the scheduled necropsy (study day 14).
Interpretation of results:
other: Toxicity category 4 for males and toxicity category 3 for females.
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
In an acute oral toxicity study with mice, performed according to OECD 423 test guideline, the LD50 of NS-1000 as a single dose in male mice is greater than 300 mg/kg bw but less than 2000 mg/kg bw (Category 4). The LD50 of NS-1000 as a single dose in female mice is greater than 50 mg/kg bw but less than 300 mg/kg bw (Category 3).


Executive summary:

The acute oral toxicity of NS-1000 was determined in accordance with OECD 423 guideline and according to GLP principles.

NS-1000 was administered to male and female mice by a single dose of 2000, 300 and 50 mg/kg bodyweight. Four of 6 males and 4/6 females at 2000 mg/kg bw and 1/6 males and 3/3 females at 300 mg/kg bw were either found dead or euthanized in extremis. All early deaths were observed between study days 2 through 8. All other animals survived to the scheduled necropsy (study day 14).

Clinical observations limited to the early death animals in the 2000 mg/kg bw group consisted of head tilt, tremors, and wet red and clear material around the mouth (post mortem). Cool body or extremities and ataxia were limited to the 300 and 2000 mg/kg bw group early death animals. Dermal atonia was limited to the 300 mg/kg bw early death animals. Other clinical observations noted at 300 and 2000 mg/kg bw, including in animals that survived to study termination, consisted of hypoactivity, decreased defecation, poor body condition (water bottle added), and partial closure of the eye(s). For the animals that survived to the scheduled necropsy, a blue abdomen was observed in the 300 mg/kg bw group males and body weight loss was noted in the 50, 300, and 2000 mg/kg bw groups. Most of the aforementioned clinical observations occurred between study days 1 and 8.

Three of the 5 surviving males in the 300 mg/kg bw group and both surviving males in the 2000 mg/kg bw group lost weight during the first week after dosing (study days 0 to 7) but then gained weight during the second week after dosing (study days 7 to 14). All surviving males surpassed their study day 0 body weight at the end of the 14-day observation period with the exception of 2000 mg/kg bw group one male. The 6 surviving females in the 50 mg/kg bw group lost a small amount of weight during the first or second week after dosing. Both surviving females in the 2000 mg/kg bw group lost weight during the first week after dosing but then gained weight during the second week after dosing (study days 7 to 14). Three of the surviving females in the 50 mg/kg bw group and both surviving females in the 2000 mg/kg bw group surpassed their study day 0 body weight at the end of the 14-day observation period.

Macroscopic findings for early death animals included a small thymus for 3 males and 2 females at 2000 mg/kg bw, a small spleen for 2 males and 2 females at 2000 mg/kg bw, and dark red discoloration of the lungs for 2 females at 300 mg/kg bw and 1 male at 2000 mg/kg bw. In addition, 1 female that was euthanized in extremis at 300 mg/kg bw had dark red areas in the glandular portion of the stomach and dark red contents in the duodenum and jejunum. One female at 300 mg/kg bw that died had dark red contents in the stomach. There were no gross findings for the animals that survived to the scheduled necropsy (study day 14).

The oral LD50 of NS-1000 as a single dose in male mice is greater than 300 mg/kg bw but less than 2000 mg/kg bw (Category 4). The oral LD50 of NS-1000 as a single dose in female mice is greater than 50 mg/kg bw but less than 300 mg/kg bw (Category 3).

Based on the effects observed in the female mice, NS-1000 shall be classified for acute oral toxicity Category 3 and shall be labelled with H301: Toxic if swallowed, according to Regulation (EC) No 1272/2008 on classification, labelling and packaging.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
discriminating dose
Value:
300 mg/kg bw
Quality of whole database:
The study has a klimisch code 1.

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Acute oral rat:

The acute oral toxicity of NS-1000 was determined in accordance with OECD 423 guideline and according to GLP principles.

NS-1000 was administered to three male and three female rats by a single dose of 2000 mg/kg bodyweight. All animals survived to the scheduled necropsy. Clinical observations were limited to dried and/or wet yellow material on the urogenital and/or anogenital areas for 1 female on 1 to 3 occasions during study days 1 to 4. There were no test substance-related body weight changes noted during the study. All rats gained weight at each interval with the exception of 1 female that had a slight (8 grams) body weight loss for study days 0 to 7. This isolated body weight loss was not attributed to NS-1000 administration. There were no macroscopic findings at the scheduled necropsy. Based on the results of this study, the estimated LD50 of NS-3000 was greater than 2000 mg/kg body weight and the substance does not need to be classified for acute oral toxicity according to Regulation (EC) No 1272/2008 on classification, labelling and packaging.

Acute oral mouse:

The acute oral toxicity of NS-1000 was determined in accordance with OECD 423 guideline and according to GLP principles.

NS-1000 was administered to male and female mice by a single dose of 2000, 300 and 50 mg/kg bodyweight. Four of 6 males and 4/6 females at 2000 mg/kg bw and 1/6 males and 3/3 females at 300 mg/kg bw were either found dead or euthanized in extremis. All early deaths were observed between study days 2 through 8. All other animals survived to the scheduled necropsy (study day 14).

Clinical observations limited to the early death animals in the 2000 mg/kg bw group consisted of head tilt, tremors, and wet red and clear material around the mouth (post mortem). Cool body or extremities and ataxia were limited to the 300 and 2000 mg/kg bw group early death animals. Dermal atonia was limited to the 300 mg/kg bw early death animals. Other clinical observations noted at 300 and 2000 mg/kg bw, including in animals that survived to study termination, consisted of hypoactivity, decreased defecation, poor body condition (water bottle added), and partial closure of the eye(s). For the animals that survived to the scheduled necropsy, a blue abdomen was observed in the 300 mg/kg bw group males and body weight loss was noted in the 50, 300, and 2000 mg/kg bw groups. Most of the aforementioned clinical observations occurred between study days 1 and 8.

Three of the 5 surviving males in the 300 mg/kg bw group and both surviving males in the 2000 mg/kg bw group lost weight during the first week after dosing (study days 0 to 7) but then gained weight during the second week after dosing (study days 7 to 14). All surviving males surpassed their study day 0 body weight at the end of the 14-day observation period with the exception of 2000 mg/kg bw group one male. The 6 surviving females in the 50 mg/kg bw group lost a small amount of weight during the first or second week after dosing. Both surviving females in the 2000 mg/kg bw group lost weight during the first week after dosing but then gained weight during the second week after dosing (study days 7 to 14). Three of the surviving females in the 50 mg/kg bw group and both surviving females in the 2000 mg/kg bw group surpassed their study day 0 body weight at the end of the 14-day observation period.

Macroscopic findings for early death animals included a small thymus for 3 males and 2 females at 2000 mg/kg bw, a small spleen for 2 males and 2 females at 2000 mg/kg bw, and dark red discoloration of the lungs for 2 females at 300 mg/kg bw and 1 male at 2000 mg/kg bw. In addition, 1 female that was euthanized in extremis at 300 mg/kg bw had dark red areas in the glandular portion of the stomach and dark red contents in the duodenum and jejunum. One female at 300 mg/kg bw that died had dark red contents in the stomach. There were no gross findings for the animals that survived to the scheduled necropsy (study day 14).

The oral LD50 of NS-1000 as a single dose in male mice is greater than 300 mg/kg bw but less than 2000 mg/kg bw (Category 4). The oral LD50 of NS-1000 as a single dose in female mice is greater than 50 mg/kg bw but less than 300 mg/kg bw (Category 3).


Justification for selection of acute toxicity – oral endpoint
Effects were observed in the OECD 423 study performed with male and female mice.

Justification for classification or non-classification

Based on the effects observed in the female mice, NS-1000 shall be classified for acute oral toxicity Category 3 and shall be labelled with H301: Toxic if swallowed, according to Regulation (EC) No 1272/2008 on classification, labelling and packaging.