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Description of key information

Acute oral toxicity of Scentaurus Clean (GR-86 -6599):

Assessment of acute oral toxicity with GR-86-6599 in the rat (Acute Toxic Class Method).

The study was carried out based on the guidelines described in:

- OECD No.423 (2001) "Acute Oral Toxicity, Acute Toxic Class Method"

- Commission Regulation (EC) No 440/2008, B1 tris: "Acute Oral Toxicity, Acute Toxic Class Method"

- EPA, OPPTS 870.1100 (2002), "Acute Oral Toxicity" JMAFF Guidelines (2000), including the most recent revisions.

The oral LD50 value of GR-86-6599 in Wistar rats was established to exceed 2000 mg/kg body weight.

According to the OECD 423 test guideline, the LD50 cut-off value was considered to exceed 5000 mg/kg body weight as no mortality occurred.

Acute inhalation toxicity of Scentaurus Clean (GR-86 -6599):

The study was carried out based on the guidelines described in:

• OECD Guidelines, Section 4, Health Effects. No.403, "Acute Inhalation Toxicity", Sep 2009.

• Commission Regulation (EC) No 440/2008, B.2. Acute Toxicity (inhalation), L142, May 2008.

• EPA OPPTS 870.1300, Acute inhalation Toxicity. EPA 712-C-98-193, August 1998.

• JMAFF Guidelines (2000), including the most recent revisions.

The inhalation LC50, 4h value of GR-86-6599 in Wistar rats was established to be within the range of 1 – 5 mg/L.

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:
17 November to 14 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
conducted under GLP conditions
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
2001
Deviations:
yes
Remarks:
Deviations from the minimum level of daily mean relative humidity occurred and the first group of 3 animals were deprived of food overnight at the end of Day 1. These deviations did not affect the outcome of the study.
Qualifier:
according to guideline
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Deviations:
yes
Remarks:
Deviations from the minimum level of daily mean relative humidity occurred and the first group of 3 animals were deprived of food overnight at the end of Day 1. These deviations did not affect the outcome of the study.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Version / remarks:
2002
Deviations:
yes
Remarks:
Deviations from the minimum level of daily mean relative humidity occurred and the first group of 3 animals were deprived of food overnight at the end of Day 1. These deviations did not affect the outcome of the study.
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
Species:
Rat, Wistar strain Crl:WI (Han) (outbred, SPF-Quality).
Recognized by international guidelines as the recommended test system (e.g. OECD, EC).
Source: Charles River Deutschland, Sulzfeld, Germany.

Number of animals:
6 Females (nulliparous and non-pregnant). Each dose group consisted of 3 animals.

Age and body weight:
Young adult animals (approx. 11-12 weeks old) were selected.
Body weight variation did not exceed +/- 20% of the sex mean.

Identification:
Earmark and tail mark

Health inspection:
At least prior to dosing. It was ensured that the animals were healthy and without any abnormality that might have affected the study integrity.

Conditions:
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative
humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle.
Any variations to these conditions were maintained in the raw data and had no effect on the
outcome of the study.

Accommodation:
Group housing of 3 animals per cage in labeled Makrolon cages (MIV type; height 18 cm.)
containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier &
Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri,
Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
Acclimatization period was at least 5 days before start of treatment under laboratory
conditions.

Diet:
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest,
Germany).

Water:
Free access to tap water.
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was
performed according to facility standard procedures. There were no findings that could
interfere with the study.
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Oral gavage, using plastic feeding tubes. The test item was stirred on a magnetic stirrer during dosing.
One single dosage on day 1.
Animals were deprived of food overnight prior to dosing and until 3-4 hours after administration of the test item. Water was available ad libitum.
Doses:
2000 mg/kg (2.17 mL/kg) body weight.
Dose volume calculated as dose level (g/kg) / specific gravity.
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
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 2000 mg/kg. 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.

Observations:
- Mortality/Viability: Twice daily
- 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. The signs were graded according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 4: grading slight (1) to very severe (4)
Maximum grade 3: grading slight (1) to severe (3)
Maximum grade 1: presence is scored (1).
- Necropsy: At the end of the observation period, all animals were sacrificed by oxygen/carbon dioxide procedure and subjected to necropsy. Descriptions of all internal macroscopic abnormalities were recorded.
Statistics:
No statistical analysis was performed (The method used is not intended to allow the calculation of a precise LD50 value).
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Key result
Sex:
female
Dose descriptor:
LD50 cut-off
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality occurred.
Clinical signs:
Hunched posture and piloerection were noted for all animals on Days 1 and/or 2.
Body weight:
The mean body weight gain shown by the 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 of the animals.
Interpretation of results:
GHS criteria not met
Conclusions:
The oral LD50 value of GR-86-6599 in Wistar rats was established to exceed 2000 mg/kg body weight.
According to the OECD 423 test guideline, the LD50 cut-off value was considered to exceed 5000 mg/kg body weight.
Based on these results, GR-86-6599 does not have to be classified and has no obligatory labelling requirement for acute oral toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).
Executive summary:

Assessment of acute oral toxicity with GR-86-6599 in the rat (Acute Toxic Class Method).

The study was carried out based on the guidelines described in:

- OECD No.423 (2001) "Acute Oral Toxicity, Acute Toxic Class Method"

- Commission Regulation (EC) No 440/2008, B1 tris: "Acute Oral Toxicity, Acute Toxic Class Method"

- EPA, OPPTS 870.1100 (2002), "Acute Oral Toxicity" JMAFF Guidelines (2000), including the most recent revisions.

GR-86-6599 was administered by oral gavage to two consecutive groups of three female Wistar rats at 2000 mg/kg body weight. Animals were subjected to daily observations and weekly determination of body weight. Macroscopic examination was performed after terminal sacrifice (Day 15).

No mortality occurred.

Hunched posture and piloerection were noted for all animals on Days 1 and/or 2.

The mean body weight gain shown by the animals over the study period was considered to be normal.

No abnormalities were found at macroscopic post mortem examination of the animals.

The oral LD50 value of GR-86-6599 in Wistar rats was established to exceed 2000 mg/kg body weight.

According to the OECD 423 test guideline, the LD50 cut-off value was considered to exceed 5000 mg/kg body weight.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
5 000 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:
17 November to 28 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
conducted under GLP conditions
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
2009
Deviations:
yes
Remarks:
For the 5 mg/L exposure group, no clinical observations and body weights were recorded on Day 2. Evaluation: Sufficient data was available to warrant the study integrity. The study integrity was not adversely affected by the deviation.
Qualifier:
according to guideline
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
yes
Remarks:
For the 5 mg/L exposure group, no clinical observations and body weights were recorded on Day 2. Evaluation: Sufficient data was available to warrant the study integrity. The study integrity was not adversely affected by the deviation.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Version / remarks:
1998
Deviations:
yes
Remarks:
For the 5 mg/L exposure group, no clinical observations and body weights were recorded on Day 2. Evaluation: Sufficient data was available to warrant the study integrity. The study integrity was not adversely affected by the deviation.
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Species: Rat: Crl:WI(Han) (outbred, SPF-Quality) Recognized by international guidelines as the recommended test system (e.g. OECD, EC).
Source: Charles River Deutschland, Sulzfeld, Germany.

- Number of animals: 5 males and 5 females (females were nulliparous and non-pregnant) for the first exposure level and 5 males for the second exposure level.

- Age and body weight: Young adult animals were selected (approximately 9 weeks old). Animals used within the study were of approximately the same age and body weight variation did not exceed +/- 20% of the sex mean.

- Identification: Individual unique number by tattoo on hind leg.

- Health inspection: At least prior to exposure. It was ensured that the animals were healthy and without any abnormality that might affect the study integrity.

- Conditions:
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

- Accommodation:
Group housing of five animals per sex per cage in labelled Makrolon cages (type IV; height 18 cm) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom). Acclimatization period was at least 5 days before start of treatment under laboratory conditions.

- Diet:
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) except during exposure to the test item.

- Water:
Free access to tap water except during exposure to the test item.

- Animal husbandry on the day of exposure:
The animals were moved to the inhalation area to in order to perform the exposure. During the exposure, there was no access to food and water. After exposure, the animals were returned to their cages which were placed in a fume cupboard for a short time period to allow test item remnants to evaporate. A sheet of filter paper was used to cover the bedding material to prevent suffocation in case of bad health condition and in order to recover and to aid the clinical observations. The sheet was removed and before the end of the exposure day, the surviving animals were returned to the animal room.

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 2.9 - <= 3.1 µm
Geometric standard deviation (GSD):
>= 1.9 - <= 2.4
Remark on MMAD/GSD:
The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined twice during the exposure period.
Details on inhalation exposure:
aerosol and nose only

Target concentrations were based on the cut off concentration values specified in the UN and EC classification guidelines. Five animals of each sex were exposed in a limit test for 4 hours to a target concentration of the test item of 5 mg/L. Based on the results, the males were identified as most sensitive sex and therefore one additional group of five males was exposed to the next lower target concentration of 1 mg/L.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
5mg/l and 1mg/l
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
Assessment of acute inhalation toxicity with GR-86-6599 in the rat (nose-only)
The study was carried out based on the guidelines described in:
• OECD Guidelines, Section 4, Health Effects. No.403, "Acute Inhalation Toxicity", Sep 2009.
• Commission Regulation (EC) No 440/2008, B.2. Acute Toxicity (inhalation), L142, May 2008.
• EPA OPPTS 870.1300, Acute inhalation Toxicity. EPA 712-C-98-193, August 1998.
• JMAFF Guidelines (2000), including the most recent revisions.
GR-86-6599 was administered as an aerosol by nose-only inhalation for 4 hours to two groups of five male and five female Wistar rats. Mortality and clinical signs were observed daily during the observation period and body weights were determined on Days 1, 2, 4, 8 and 15 and at death. Macroscopic examination was performed on the day of death or after terminal sacrifice (Day 15).
For the 5 mg/L exposure group, the time-weighted mean actual concentration was 5.1 ± 0.1 mg/L. The nominal concentration (amount of test item used divided by the volume of pressurized air used) was 6.9 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 74%. For the 1 mg/L exposure group, the time-weighted mean actual concentration was 1.2 ± 0.03 mg/L. The nominal concentration (amount of test item used divided by the volume of pressurized air used) was 1.7 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 67%. The concentration measurements equally distributed over time showed that the item was sufficiently stable.
The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined twice during the exposure period. At 5 mg/L, the MMAD was 3.0 μm (gsd 1.9) and 3.0 μm (gsd 2.1). At 1 mg/L, the MMAD was 2.9 μm (gsd 2.0) and 3.1 μm (gsd 2.4)
Statistics:
No statistical analysis was performed.
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 1 - < 5 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
At 5 mg/L, one male was found dead and one male and one female were sacrificed for humane reasons on Day 3. Two males were sacrificed between Days 7 and 9. No further mortality occurred in any of the other animals assigned to the study
Clinical signs:
other: After exposure at 5 mg/L, lethargy, hunched posture, slow breathing, labored respiration, rales, piloerection and ptosis up to Day 9. Scaleson the back of the females - Days 10 and 13. After exposure at 1 mg/L, hunched posture and rales - Days 1 and 4.
Body weight:
At 5 mg/L, body weight loss was noted for all surviving animals during the first week post exposure. All animals regained weight during the second week.
At 1 mg/L, the body weight gain shown by the animals over the study period was within the range expected for rats of this strain and age used in this type of study and was therefore considered not indicative of toxicity.
Gross pathology:
sacrificed for ethical reasons during the study revealed abnormalities of the lungs (pale with many dark red or black), stomach and intestines (distended with gas) and thymus (many dark red foci). No abnormalities were seen for the surviving animals.
At 1 mg/L, no abnormalities were found at macroscopic examination of the animals.
Incidental findings included advanced autolysis for one male found dead (5 mg/L). This finding was toxicologically not relevant.
Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
The inhalation LC50, 4h value of GR-86-6599 in Wistar rats was established to be within the range of 1 – 5 mg/L.
Executive summary:

Assessment of acute inhalation toxicity with GR-86-6599 in the rat (nose-only)

The study was carried out based on the guidelines described in:

• OECD Guidelines, Section 4, Health Effects. No.403, "Acute Inhalation Toxicity", Sep 2009.

• Commission Regulation (EC) No 440/2008, B.2. Acute Toxicity (inhalation), L142, May 2008.

• EPA OPPTS 870.1300, Acute inhalation Toxicity. EPA 712-C-98-193, August 1998.

• JMAFF Guidelines (2000), including the most recent revisions.

GR-86-6599 was administered as an aerosol by nose-only inhalation for 4 hours to two groups of five male and five female Wistar rats. Mortality and clinical signs were observed daily during the observation period and body weights were determined on Days 1, 2, 4, 8 and 15 and at death. Macroscopic examination was performed on the day of death or after terminal sacrifice (Day 15).

For the 5 mg/L exposure group, the time-weighted mean actual concentration was 5.1 ± 0.1 mg/L. The nominal concentration (amount of test item used divided by the volume of pressurized air used) was 6.9 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 74%. For the 1 mg/L exposure group, the time-weighted mean actual concentration was 1.2 ± 0.03 mg/L. The nominal concentration (amount of test item used divided by the volume of pressurized air used) was 1.7 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 67%. The concentration measurements equally distributed over time showed that the item was sufficiently stable.

The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined twice during the exposure period. At 5 mg/L, the MMAD was 3.0 μm (gsd 1.9) and 3.0 μm (gsd 2.1). At 1 mg/L, the MMAD was 2.9 μm (gsd 2.0) and 3.1 μm (gsd 2.4)

At 5 mg/L, one male was found dead and one male and one female were sacrificed for humane reasons on Day 3. Two males were sacrificed between Days 7 and 9. No further mortality occurred in any of the other animals assigned to the study.

At 5 mg/L, slow respiration was noted during exposure. After exposure, lethargy, hunched posture, slow breathing, labored respiration, rales, piloerection and ptosis were seen for the animals up to Day 9. Scales were seen on the back of the females between Days 10 and 13. At 1 mg/L, no abnormalities were noted during exposure. After exposure, hunched posture and rales were seen for the animals between Days 1 and 4.

At 5 mg/L, body weight loss was noted for all surviving animals during the first week post exposure. All animals regained weight during the second week. At 1 mg/L, the body weight gain shown by the animals over the study period was considered not indicative of toxicity.

At 5 mg/L, macroscopic post mortem examination of the animals that were found dead or sacrificed for ethical reasons during the study revealed abnormalities of the lungs (pale with many dark red or black), stomach and intestines (distended with gas) and thymus (many dark red foci). No abnormalities were seen for the surviving animals. At 1 mg/L, no abnormalities were found at macroscopic examination of the animals.

The inhalation LC50, 4h value of GR-86-6599 in Wistar rats was established to be within the range of 1 – 5 mg/L.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LC50
Value:
1 000 000 µg/m³
Quality of whole database:
between 1 and 5 mg/L

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because inhalation of the substance is likely
Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
The study does not need to be conducted because inhalation of the substance is likely

Additional information

Justification for classification or non-classification

Based on the acute oral toxicity study, Scentaurus Clean (GR-86-6599) does not have to be classified and has no obligatory labelling requirement for acute oral toxicity (LD50 > 5000 mg/kg) according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Based on the acute inhalation toxicity study, Scentaurus Clean (GR-86 -6599) has to be classified as Acute Toxicity Inhalation category 4 (LC50 < 5 mg/L) according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

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