5,9-dimethyldec-4-enal

Endpoint summary

Description of key information

Oral: LD50 > 300 - < 2000 mg/kg bw, female rat, OECD TG 420, 2016

Inhalation: LC50 (female): 3.00 (C.I. 1.50 – 4.50) mg/L, OECD TG 403, 2016

Dermal: measured LD50 > 2000 mg/kg bw and the estimated LD50 cut-off value was considered to be > 5000 mg/kg bw, male/female rat, OECD TG 402, 2016

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:

10-12-2015 to 28-01-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Justification for type of information:

Information as to the availability of the in vivo study is provided in 'attached justification'.

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)

Remarks:

inspected: June 2015; signature: September 2015

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
- Source: Recognised supplier
- Age at study initiation: 8 - 12 weeks.
- Weight at study initiation: 177 - 195 g (300 mg/kg including sighting test; sentinel); 190 - 211 g (2000 mg/kg sighting test; sentinel); The weight variation did not exceed ±20% of the mean weight during the test.
- Fasting period before study: Overnight before dosing and three to four hours after dosing.
- Housing: Group housed in groups of up to four in suspended solid-floor polypropylene cages furnished with woodflakes.
- Diet (e.g. ad libitum): Certified diet from recognised supplier, provided ad libitum (except for fasting period).
- Water (e.g. ad libitum): ad libitum (except for fasting period)
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70%
- Air changes (per hr): > 15 air changes per hour
- Photoperiod: 12 h light / 12 h dark

IN-LIFE DATES: From: To: 2015-12-10 to 2016-01-28

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

The specific gravity was determined and used to calculate the appropriate dose volume for the required dose level.
For the purpose of the 300 mg/kg dose level the test item was freshly prepared, as required, as a solution in arachis oil BP. Arachis oil BP was used because the test item did not dissolve/suspend in distilled water. For the purpose of the 2000 mg/kg dose level, the test item was used as supplied. The 300 mg/kg bw and 2000 mg/kg bw dose levels were treated stepwise. Singuarly and in the absence of mortality or evident toxicity a further group of 4 was tested in the appropriate dose level.

Doses:

300 mg/kg bw (initial sighting test and main study)
2000 mg/kg bw (initial sighting test and main study)

No. of animals per sex per dose:

1 (sighting study) and 4 (main study) as applicable; total 5 per dose - based on guideline specified sequential testing strategy

Control animals:

no

Details on study design:

- 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 body weights were recorded on Day 0 (the day of dosing) and on Days 7 and 14.
- Necropsy of survivors performed: yes

Sex:

female

Dose descriptor:

LD50

Effect level:

> 300 - < 2 000 mg/kg bw

Based on:

test mat.

Mortality:

2000 mg/kg bw (sentinel and follow-on test): No mortality in sentinel; 2 mortalities in definitive test
300 mg/kg bw (sentinel and definitive test): No mortality

Clinical signs:

other: 2000 mg/kg bw (sentinel): Appeared normal throughout the study ; (follow-on test): hunched posture, tiptoe gait, pilo erection, decreased respiratory rate, labored respiration, emaciation, lethargy, pallor of the extremities and hypothermia. Surviving ani

Gross pathology:

2000 mg/kg bw (sentinel and follow-on test): Abnormalities noted at necropsy in animals subject to mortality: dark liver, dark kidneys, haemorrhage and epithelial sloughing of the gastric mucosa and haemorrhage and epithelial sloughing of the non glandular epithelium of the stomach. All surivors gave no abnormalities at necropsy.
300 mg/kg bw (sentinel and definitive test): No abnormalities were noted at necropsy.

Interpretation of results:

Category 4 based on GHS criteria

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the oral LD50 was estimated to be in the range of 300 - 2000 mg/kg bw in female Wistar rats.

Executive summary:

The study was performed according to OECD TG 420 and EU Method B.1 bis Acute Toxicity (Oral) and in accordance with GLP to assess the acute oral toxicity of the test material following a single oral administration in the female Wistar strain rat by the fixed dose method. The test substance was administered by oral gavage in an initial sighting study at 2000 mg/kg bw. No mortality and no significant toxicity was observed. A further group of four fasted females was given a single oral dose of test item at a dose level of 2000 mg/kg body weight. Two mortalities were observed. Abnormalities noted at a dose level of 2000 mg/kg, within mortality related necropsy were: dark liver, dark kidneys, haemorrhage and epithelial sloughing of the gastric mucosa and haemorrhage and epithelial sloughing of the non glandular epithelium of the stomach. No abnormalities were observed in surviving animals. Due to mortality at a dose level of 2000 mg/kg, a further sighting test was performed at a dose level of 300 mg/kg. This was then followed by a further group of four fasted females at a dose level of 300 mg/kg body weight. There was no mortality, systemic toxicity or abnormal findings at necropsy and all animals gained body weight. Under the conditions of this study, the oral LD50 was estimated to be in the range of 300 - 2000 mg/kg bw in female Wistar rat. The test substance was classified as Acute Oral Toxicity: Category 4 according to Regulation (EC) 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

discriminating dose

Value:

300 mg/kg bw

Quality of whole database:

The available information as a whole meets the tonnage driven information requirements of REACH.

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:

21-06-2016 to 29-09-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met. Minor deviation: Three males Group 3 were slightly outside ±20% (lower than) of the mean body weights of the Group 1 or Group 2 males. This was not considered to impact the validity of the study.

Justification for type of information:

Information as to the availability of the in vivo study is provided in 'attached justification'.

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

yes

Remarks:

Three males Group 3 were slightly outside ±20% (lower than) of the mean body weights of the Group 2 males. Not considered to impact the validity of the study.

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Deviations:

yes

Remarks:

See above

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: June 2015; signature: September 2015

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: 8 - 12 weeks; females were nulliparous and non pregnant.
- Weight at study initiation: 200 - 350 g
- Fasting period before study: None.
- Housing: Housed in groups of up to five by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes provided with environmental enrichment items.
- Diet (e.g. ad libitum): Certified diet from recognised supplier, provided ad libitum (except for exposure period period)
- Water (e.g. ad libitum): ad libitum (except for exposure period period)
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70%
- Air changes (per hr): > 15 air changes per hour
- Photoperiod: 12 h light / 12 h dark

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks:

Oxygen concentration (%) was monitored during the study by an electronic oxygen analyzer. The test atmospheres were generated to contain at least 19% oxygen.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The test item was aerosolized using a metal concentric jet nebulizer located at the top of the exposure chamber. The nebulizer was connected to a glass syringe attached to an infusion pump, which provided a continuous supply of test item under pressure, and to a metered compressed air supply.
- Exposure chamber volume: approximately 30 litres (dimensions: 28 cm diameter x 50 cm high)
- Method of holding animals in test chamber: Prior to the day of exposure each rat was acclimatized (for approximately 2 hours) to a tapered polycarbonate restraining tube. During the exposure period, each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber ‘O’ ring. Only the nose of each animal was exposed to the test atmosphere.
- Source and rate of air: filtered air; chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.
- Method of conditioning air: Compressed air was supplied by means of an oil free compressor and passed through a water trap and respiratory quality filters before it was introduced to the nebulizer.
- System of generating particulates/aerosols: metal concentric jet nebulizer; the concentration within the exposure chamber was controlled by adjusting the rate of the infusion pump and air flow settings into the chamber. The chamber flow rate was maintained between 50 to 60 L/min providing 100 to 120 air changes per hour. 60 L/min in group 1 and 2 and 50 L/min in group 3.
- Method of particle size determination: Particle size was determined using a cascade impactor. The device consisted of six impactors stages. The mean amount for each stage was used to determine the cumulative amount below each cut-off point size to calculate the proportional (%) aerosol of defined size ranges. The resulting values were converted to probits and plotted against Log10 cut-point size. From this plot, the Mass Median Aerodynamic Diameter (MMAD) was determined (as the 50% point) and the geometric standard deviation was calculated. In addition the proportion (%) of aerosol less than 4 μm (considered to be the inhalable fraction) was determined.
- Treatment of exhaust air: The extract from the exposure chamber passed through a ‘scrubber’ trap and was connected with a high efficiency filter to a metered exhaust system. The chamber was maintained under negative pressure. A schematic of the dynamic (continuous flow) system is presented in the full study report.
- Temperature, humidity, in air chamber: The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter: 19 to 20 °C and 30 to 70%.

TEST ATMOSPHERE
- Brief description of analytical method used: The test atmosphere was sampled nine times during the exposure period. The sampling procedure involved 2 to 10 litres (dependent on target concentration) of test atmosphere being drawn through a glass fibre filter. Each filter was then submitted for chemical analysis by gas chromatography (GC). The test filter samples received were extracted with methanol to achieve the working concentration. Blank filters were accurately fortified with known amounts of test item and either analysed without further procedure or purged with nitrogen prior to analysis. A range of standard solutions were prepared in methanol from a stock solution of 1.084 mg/mL by serial dilution covering the concentration range 0.0542 to 0.1626 mg/mL. Full details of the analytical method are provided in the full study report. The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. The regression coefficients calculated were found to be 0.9999. The fortified samples of filters were found to have a recovery value of ± 10% of the fortification. The results indicate the accurate use of the test item and filters during this study. Working solutions of the test item were shown to be sufficiently stable to complete the analysis without deterioration of the analyte. Full details of the analytical method are provided in the full study report.
- Samples taken from breathing zone: yes

TEST ATMOSPHERE
- Particle size distribution: The particle size of the generated atmosphere inside the exposure chamber was determined three times during each exposure period using a cascade impactor. The particle size distribution for each group is reported in table 1.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The Mass Median Aerodynamic Diameter (MMAD) was determined and is reported for each group in table 1.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Following an appropriate equilibration period three groups were subjected to a single exposure to the test item for a period of four hours. Based on the expected toxicity of the test item, a target concentration of 5.0 mg/L was used for the first exposure. Further concentrations were selected after consideration of the results of the previous exposure. Full details are provided in table 2.

No. of animals per sex per dose:

5 per sex per dose

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days (for groups 1, 2 and 3); for group 3 the observation period was extended to day 28 so as to assess full recovery.
- Frequency of observations and weighing: Clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, one hour after termination of exposure and subsequently once daily for up to fourteen days. Any evidence of mortality or overt toxicity was recorded at each observation. Individual body weights were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 3, 7 and 14 or after mortality. Group 3 was also examined once daily for observations on days 15 - 28 and bodyweights taken on days 21 and 28 during the extended recovery period.
- Necropsy of survivors performed: yes (and in the event of any mortalities) for suvivors on day 14 and/or day 28 (group 3).
- Other examinations performed: clinical signs, body weight,organ weights, and any other relevant toxicological effects were reported.

Statistics:

Using the mortality data obtained, an estimate of the acute inhalation median lethal concentration (LC50) was calculated using validated data analysis software which utilized Log-Logistic (Logit) regression models in order to calculate the LC50 values. LC50 values and 95% confidence limits were calculated for males and females separately.

Sex:

male/female

Dose descriptor:

LC50

Effect level:

3.05 mg/L air

Based on:

test mat.

95% CL:

> 2.9 - < 3.2

Exp. duration:

4 h

Remarks on result:

other: Effect levels based on analytically confirmed: mean achieved concentrations for each exposure/sex groups

Sex:

male

Dose descriptor:

LC50

Effect level:

3.09 mg/L air

Based on:

test mat.

95% CL:

> 0.78 - < 5.41

Exp. duration:

4 h

Remarks on result:

other: Effect levels based on analytically confirmed: mean achieved concentrations for each exposure/sex groups

Sex:

female

Dose descriptor:

LC50

Effect level:

3 mg/L air

Based on:

test mat.

95% CL:

> 1.5 - < 4.5

Exp. duration:

4 h

Remarks on result:

other: Effect levels based on analytically confirmed: mean achieved concentrations for each exposure/sex groups

Mortality:

Mortalities are reported in table 3.

Clinical signs:

other: Common abnormalities noted during the study included decreased respiratory rate, sneezing, lethargy, red/brown staining around the snout, hunched posture, pilo-erection and wet fur. There were frequent instances of labored respiration, occasional instanc

Body weight:

In group 2, 1.04 mg/L: All males and females exhibited body weight losses on Day 1 post-exposure. All males exhibited body weight gains during the remainder of the recovery period with the exception of two males which showed a slightly reduced gain from Days 1 to 3 post-exposure. These were in the ranges expected and considered normal for this type of study. One female exhibited a body weight loss from Days 3 to 7 post-exposure and two females exhibited body weight losses during the final week of the recovery period.
In group 3, 3.05 mg/L: All males and females exhibited body weight losses on Day 1 post-exposure. All male survivors exhibited body weight gains during the remainder of the exposure period, with the exception of one male that showed a slightly reduced gain from days 3 to 7 of the recovery period. These were in the ranges expected and considered normal for this type of study. One female exhibited a body weight loss from Days 3 to 7 post-exposure. Both surviving females exhibited body weight losses or showed no body weight gain from Days 7 to 14 post-exposure. One of these females exhibited a further body weight loss from Days 14 to 21 post-exposure. Both surviving females exhibited body weight gains during the final week of the extended recovery period.

Gross pathology:

In group 2, 1.04 mg/L: No macroscopic abnormalities in three females; dark patches in lungs was seen in five males and two females.
In group 3, 3.05 mg/L: No macroscopic abnormalities in in three male survivors; dark patches in lungs was seen in two female survivors. In the mortalities, gaseous distension of the stomach, small and/or large intestine and/or dark patches in lung was observed.
In group 1: 5.00 mg/L: In the mortalities, gaseous distension of the stomach, small and/or large intestine and/or dark patches in lung and/or liver - dark was observed at necropsy.

Other findings:

- Other observations: The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity during necropsy.

Table 1. Characteristics of the achieved atmosphere:

Group Number	Mean Achieved Atmosphere Concentration (mg/L)	Mean Mass Median Aerodynamic Diameter (µm)	Inhalable Fraction (% <4 µm)	Geometric Standard Deviation
1	5.00	2.25	76.7	2.21
2	1.04	2.19	77.8	2.21
3	3.05	2.17	78.2	2.21

 

Table 2. Mean achieved atmosphere concentrations:

Group Number	Atmosphere Concentration
	Mean Achieved (mg/L)	Standard Deviation	Nominal (mg/L)
1	5.00	0.14	13.1
2	1.04	0.07	2.80
3	3.05	0.09	8.72

 

Table 3. Mortality data summary:

Group Number	Mean Achieved Atmosphere Concentration (mg/L)	Deaths
		Male	Female	Total
1	5.00	5/5	5/5	10/10
2	1.04	0/5	0/5	0/10
3	3.05	2/5	3/5	5/10

Interpretation of results:

Category 4 based on GHS criteria

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the inhalation LC50 (male/female) was: 3.05 (C.I. 2.90 – 3.20) mg/L; LC50 (male): 3.09 (C.I. 0.78 – 5.41) mg/L and LC50 (female): 3.00 (C.I. 1.50 – 4.50) mg/L within the RCCHan WIST rat.

Executive summary:

The study was performed according to OECD TG 403 and EU Method B.2 in accordance with GLP to assess the acute inhalation toxicity of the test item. Three groups of ten RccHanTM : WIST strain rats (five males and five females) were exposed to an aerosol atmosphere of the test item. The groups were exposed for four hours using a nose only exposure system, followed by a fourteen day observation period (group 1 and 2) or a twenty-eight day extended observation period (group 3). The mean achieved atmosphere concentrations were as follows: Group 1: 5.00 mg/L, Group 2: 1.04 mg/L and Group 3: 3.05 mg/L. The characteristics of the achieved atmosphere where Mean Mass Median Diameter (particle size) and Inhalable Fraction <4 μm: Group 1: 2.25 μm and 76.7%; Group 2: 2.19 μm and 77.8% and Group 3: 2.17 μm and 78.2%. The Geometric Standard Deviation was 2.21 in all groups, respectively. There was 5 male and 5 female mortalities in Group 1, no mortalities in Group 2 and 2 male and 3 female mortalities in Group 3. Within Group 2, all males and females exhibited body weight losses or showed no body weight gain on Day 1 post-exposure. All males exhibited body weight gains during the remainder of the recovery period with the exception of two males which showed a slightly reduced gain from Days 1 to 3 post-exposure. These were in the ranges expected and considered normal for this type of study. One female exhibited a body weight loss from Days 3 to 7 post-exposure and two females exhibited body weight losses during the final week of the recovery period. Applicant assessment indicated these were not significant as they were not combined with clinical signs or abnormal necropsy. Within Group 3, all surviving males and females exhibited body weight losses on Day 1 post-exposure. All male survivors exhibited body weight gains during the remainder of the exposure period, with the exception of one male that showed a slightly reduced gain from days 3 to 7 of the recovery period. These were in the ranges expected and considered normal for this type of study. One female exhibited a body weight loss from Days 3 to 7 post-exposure. Both surviving females exhibited body weight losses or showed no body weight gain from Days 7 to 14 post-exposure. One of these females exhibited a further body weight loss from Days 14 to 21 post-exposure. Both surviving females exhibited body weight gains during the final week of the extended recovery period. In Group 2 clinical observations for all males and females appeared normal at day 2 post exposure. In Group 3 survivors (male/female) recovered such that no significant observations were apparent on Day 5 post-exposure. Three surviving males indicated a deterioration in condition exhibiting observations again from Day 6, subsequently, the surviving males appeared normal on Day 9 post-exposure, however two of these males deteriorated slightly on Day 20 (decreased respiratory rate) but subsequently appeared normal again on Day 26. Two surviving females appeared normal from Days 8 or 9 post-exposure but showed a deterioration in condition on Day 11 with observations persisting in one of these females until the end of the extended recovery period (decreased respiratory rate and laboured respiration). One other surviving female appeared normal for one day (Day 14) and then on Days 27 and 28 post-exposure. During necropsy in Group 2 no macroscopic abnormalities were seen in three females; dark patches in lungs was seen in five males and two females. In Group 3, no macroscopic abnormalities were observed in in three male survivors dark patches in lungs was seen in two female survivors. In the mortalities, gaseous distension of the stomach, small and/or large intestine and/or dark patches in lung was observed. Under the conditions of this study, the inhalation LC50 (male/female) was: 3.05 (C.I. 2.90 – 3.20) mg/L; LC50 (male): 3.09 (C.I. 0.78 – 5.41) mg/L and LC50 (female): 3.00 (C.I. 1.50 – 4.50) mg/L within the RCCHan WIST rat. The test item was classified as Acute Inhalation Toxicity: Category 4 according to Regulation (EC) 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

3 000 mg/m³ air

Quality of whole database:

The available information as a whole meets the tonnage driven information requirements of REACH.

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:

11-05-2016 to 01-06-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Justification for type of information:

Information as to the availability of the in vivo study is provided in 'attached justification'.

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)

Remarks:

inspected: June 2015; signature: September 2015

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: Recognised supplier
- Age at study initiation: 8 - 12 weeks.
- Weight at study initiation: 226 - 270 g; the weight variation did not exceed ±20% of the mean weight for each sex.
- Fasting period before study: Not applicable
- Housing: suspended solid floor polypropylene cages furnished with woodflakes; 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 (e.g. ad libitum): Certified diet from recognised supplier, provided ad libitum.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70%
- Air changes (per hr): > 15 air changes per hour
- Photoperiod: 12 h light / 12 h dark

IN-LIFE DATES: From: To: 2016-05-11 to 2016-06-01

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: the day before treatment the back and flanks were clipped free of hair. Dorsal area application.
- % coverage: Approximately 10% of total body surface
- Type of wrap if used: The area of application was covered 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 wiped with cotton wool moistened with arachis oil BP to remove any residual test item
- Time after start of exposure: 24 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg
- Concentration (if solution): Not applicable.
- Constant volume or concentration used: Not applicable.

Duration of exposure:

24 hours

Doses:

2000 mg/kg

No. of animals per sex per dose:

5 per sex per dose (5 male/5 female); treated sequentially following application to 1 sentinel per sex per dose .

Control animals:

not required

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical observations and mortality checks were conducted at approximately 0.5, 1, 2, and 4 hours and subsequently once daily for 14 days. Local effects were examined once daily for 14 days after the completion of the 24-hour exposure period. Full details on the scoring and criteria (consistent with Draize) are given in the full study report. Individual bodyweights were recorded prior to application of the test item on Day 0 and on Days 7 and 14.
- Necropsy of survivors performed: yes

Statistics:

No statistical analyses were performed.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality was observed.

Clinical signs:

other: - Clinical observations: No signs of systemic toxicity were noted during the observation period. - Dermal reactions: Signs of dermal irritation noted were very slight to well defined erythema, very slight edema and light brown discoloration of the epiderm

Gross pathology:

No abnormalities were noted at necropsy.

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the dermal LD50 was established to exceed 2000 mg/kg bw in male/female Wistar rat. Under the conditions of this study and under the Globally Harmonized Classification System of Classification and Labelling of Chemicals (GHS), the LD50 cut-off value was considered to be greater than 5000 mg/kg body weight.

Executive summary:

The study was performed according to OECD TG 402 and EU Method B.3 Acute Toxicity (Dermal) and in accordance with GLP to assess the acute dermal toxicity of the test substance in the Wistar strain rat. A group of ten animals (five males and five females) was given a single, 24 hour, semi-occluded dermal application of the undiluted test item to intact skin at a dose level of 2000 mg/kg body weight. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy. There were no signs of system toxicity or abnormalities on necropsy. There was no mortality during the study. Signs of dermal irritation noted were very slight to well‑defined erythema, very slight edema and light brown discoloration of the epidermis. Which had fully reversed by day 7 in the case of irritation and day 8 in the case of discolouration. Animals showed expected gains in body weight, except for one female which showed body weight loss during the first week with expected gain in body weight during the second week. Under the conditions of this study, the dermal LD50 was established to exceed 2000 mg/kg bw in male/female Wistar rat. Under the conditions of this study, and according to the GHS criteria, the LD50 cut-off value was considered to be greater than 5000 mg/kg body weight.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

2 000 mg/kg bw

Quality of whole database:

The available information as a whole meets the tonnage driven information requirements of REACH.

Additional information

ORAL:

Key study : OECD TG 420, 2016 : The study was performed according to OECD TG 420 and EU Method B.1 bis Acute Toxicity (Oral) and in accordance with GLP to assess the acute oral toxicity of the test material following a single oral administration in the female Wistar strain rat by the fixed dose method. The test substance was administered by oral gavage in an initial sighting study at 2000 mg/kg bw. No mortality and no significant toxicity was observed. A further group of four fasted females was given a single oral dose of test item at a dose level of 2000 mg/kg body weight. Two mortalities were observed. Abnormalities noted at a dose level of 2000 mg/kg, within mortality related necropsy were: dark liver, dark kidneys, haemorrhage and epithelial sloughing of the gastric mucosa and haemorrhage and epithelial sloughing of the non glandular epithelium of the stomach. No abnormalities were observed in surviving animals. Due to mortality at a dose level of 2000 mg/kg, a further sighting test was performed at a dose level of 300 mg/kg. This was then followed by a further group of four fasted females at a dose level of 300 mg/kg body weight. There was no mortality, systemic toxicity or abnormal findings at necropsy and all animals gained body weight. Under the conditions of this study, the oral LD50 was estimated to be in the range of 300 - 2000 mg/kg bw in female Wistar rat. The test substance was classified as Acute Oral Toxicity: Category 4 according to Regulation (EC) 1272/2008.

 

INHALATION:

Key study : OECD TG 403, 2016 : The study was performed according to OECD TG 403 and EU Method B.2 in accordance with GLP to assess the acute inhalation toxicity of the test item. Three groups of ten RccHanTM : WIST strain rats (five males and five females) were exposed to an aerosol atmosphere of the test item. The groups were exposed for four hours using a nose only exposure system, followed by a fourteen day observation period (group 1 and 2) or a twenty-eight day extended observation period (group 3). The mean achieved atmosphere concentrations were as follows: Group 1: 5.00 mg/L, Group 2: 1.04 mg/L and Group 3: 3.05 mg/L. The characteristics of the achieved atmosphere where Mean Mass Median Diameter (particle size) and Inhalable Fraction <4 μm: Group 1: 2.25 μm and 76.7%; Group 2: 2.19 μm and 77.8% and Group 3: 2.17 μm and 78.2%. The Geometric Standard Deviation was 2.21 in all groups, respectively. There was 5 male and 5 female mortalities in Group 1, no mortalities in Group 2 and 2 male and 3 female mortalities in Group 3. Within Group 2, all males and females exhibited body weight losses or showed no body weight gain on Day 1 post-exposure. All males exhibited body weight gains during the remainder of the recovery period with the exception of two males which showed a slightly reduced gain from Days 1 to 3 post-exposure. These were in the ranges expected and considered normal for this type of study. One female exhibited a body weight loss from Days 3 to 7 post-exposure and two females exhibited body weight losses during the final week of the recovery period. Applicant assessment indicated these were not significant as they were not combined with clinical signs or abnormal necropsy. Within Group 3, all surviving males and females exhibited body weight losses on Day 1 post-exposure. All male survivors exhibited body weight gains during the remainder of the exposure period, with the exception of one male that showed a slightly reduced gain from days 3 to 7 of the recovery period. These were in the ranges expected and considered normal for this type of study. One female exhibited a body weight loss from Days 3 to 7 post-exposure. Both surviving females exhibited body weight losses or showed no body weight gain from Days 7 to 14 post-exposure. One of these females exhibited a further body weight loss from Days 14 to 21 post-exposure. Both surviving females exhibited body weight gains during the final week of the extended recovery period. In Group 2 clinical observations for all males and females appeared normal at day 2 post exposure. In Group 3 survivors (male/female) recovered such that no significant observations were apparent on Day 5 post-exposure. Three surviving males indicated a deterioration in condition exhibiting observations again from Day 6, subsequently, the surviving males appeared normal on Day 9 post-exposure, however two of these males deteriorated slightly on Day 20 (decreased respiratory rate) but subsequently appeared normal again on Day 26. Two surviving females appeared normal from Days 8 or 9 post-exposure but showed a deterioration in condition on Day 11 with observations persisting in one of these females until the end of the extended recovery period (decreased respiratory rate and laboured respiration). One other surviving female appeared normal for one day (Day 14) and then on Days 27 and 28 post-exposure. During necropsy in Group 2 no macroscopic abnormalities were seen in three females; dark patches in lungs was seen in five males and two females. In Group 3, no macroscopic abnormalities were observed in in three male survivors dark patches in lungs was seen in two female survivors. In the mortalities, gaseous distension of the stomach, small and/or large intestine and/or dark patches in lung was observed. Under the conditions of this study, the inhalation LC50 (male/female) was: 3.05 (C.I. 2.90 – 3.20) mg/L; LC50 (male): 3.09 (C.I. 0.78 – 5.41) mg/L and LC50 (female): 3.00 (C.I. 1.50 – 4.50) mg/L within the RCCHan WIST rat. The test item was classified as Acute Inhalation Toxicity: Category 4 according to Regulation (EC) 1272/2008.

 

References:

1. OECD TG 403 (2009)

2. OECD 39 (2009)

 

Applicant assessment indicates: no significant changes were observed at 1.04 mg/L dose level in the upper respiratory tract (such as clinical signs: dyspnoea or rhinitis and/or histopathological effects like hyperemia, oedema and inflammation) or lower respiratory tract, which may be post-mortality or termination related effects and not indicative of function changes or toxicologically relevant morphological change in the absence of mortality. There are no marked changes reported indicative of organ dysfunction sufficient for classification under Regulation (EC) 1272/2008 as STOT-SE. This conclusion is particularly in relation to no significant effects being observed in the absence of mortality at any dose level and due to the application of Acute Inhalation Toxicity: Category 4 according to Regulation (EC) 1272/2008, to the substance.

 

DERMAL:

Key study : OECD TG 402, 2016 : The study was performed according to OECD TG 402 and EU Method B.3 Acute Toxicity (Dermal) and in accordance with GLP to assess the acute dermal toxicity of the test substance in the Wistar strain rat. A group of ten animals (five males and five females) was given a single, 24 hour, semi-occluded dermal application of the undiluted test item to intact skin at a dose level of 2000 mg/kg body weight. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy. There were no signs of system toxicity or abnormalities on necropsy. There was no mortality during the study. Signs of dermal irritation noted were very slight to well‑defined erythema, very slight edema and light brown discoloration of the epidermis. Which had fully reversed by day 7 in the case of irritation and day 8 in the case of discolouration. Animals showed expected gains in body weight, except for one female which showed body weight loss during the first week with expected gain in body weight during the second week. Under the conditions of this study, the dermal LD50 was established to exceed 2000 mg/kg bw in male/female Wistar rat. Under the conditions of this study, and according to the GHS criteria, the LD50 cut-off value was considered to be greater than 5000 mg/kg body weight.

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for acute toxicity: oral category 4: H302

The substance meets classification criteria under Regulation (EC) No 1272/2008 for acute toxicity: inhalation category 4: H332

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for acute toxicity: dermal