Cyclohexanepropanol, 2,2,3,6-tetramethyl-.alpha.-propyl-

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15-11-2017 to 27-12-2017

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.

Justification for type of information:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: January 2018 ; signature: June 2018

Test type:

standard acute method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

478-330-3

EC Name:

-

Cas Number:

95851-08-4

Molecular formula:

C16H32O

IUPAC Name:

1-[(1R,3S,6S)-2,2,3,6-tetramethylcyclohexyl]hexan-3-ol

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: Dry area, protected from light, in a refrigerator (temp 2-8 °C), store under nitrogen in a closed container after first opening
- Other: colourless liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

RccHan :WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Rationale for alternative/additional species to rat (if applicable): Not applicable.
- Source: Recognised supplier (documented in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Rationale for use of males (if applicable): According to OECD TG 403 guidelines.
- Age at study initiation: 8 - 9 weeks (prior to acclimatisation) and 9 – 10 weeks at study initiation ; females were nulliparous and non pregnant.
- Weight at study initiation: 255 - 273 g (males) and 177 – 196 g (females)
- Fasting period before study: None.
- Housing: Housed in groups of up to five by sex in solid-floor polycarbonate cages with stainless steel mesh lids, furnished with softwood flakes provided with environmental enrichment items.
- Historical data: The laboratory has a historic control dataset (not documented in the full study report).
- 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:
- Microbiological status when known: No issues reported within the study.
- Method of randomisation in assigning animals to test and control groups: Randomly allocated to cages after receipt.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70%
- Air changes (per hr): typically : > 15 air changes per hour
- Photoperiod: 12 h light / 12 h dark

IN-LIFE DATES: From: 07-12-2017 To: 27-12-2017

Administration / exposure

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 analyser. The test atmospheres were generated to contain at least 19% oxygen.

Mass median aerodynamic diameter (MMAD):

2.3 µm

Geometric standard deviation (GSD):

2.6

Remark on MMAD/GSD:

Group 1: 5.0 mg/L (Target aerosol concentration) or 2.08 mg/L Mean achieved concentration. The characteristics of the achieved atmosphere where Mean Mass Median Diameter (particle size) and fraction < 7 μm: Group 1: 2.3 μm and 87%. The Geometric Standard Deviation was 2.60.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The test item was aerosolized using a metal concentric jet atomizer/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: 30 cm diameter x 45 cm high)
- Method of holding animals in test chamber: During the exposure period, each rat was individually held in a tapered, restraining tube fitted onto a single tier of the exposure chamber and sealed. 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 20 L/min providing 40 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 19 to 20 L/min providing 12 to 40 air changes per hour.
- Method of particle size determination: Particle size was determined using a cascade impactor. The device consisted of eight 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. From this plot, the Mass Median Aerodynamic Diameter (MMAD) was determined (as the 50% point) and the geometric standard deviation was calculated.
- Treatment of exhaust air: The extract from the exposure chamber passed through a filtration system and was connected to a metered exhaust system. The chamber was maintained under negative pressure (19 L/min exiting nebulizer and extract airflow at 20 L/min). A schematic of the dynamic (continuous flow) system is presented in the full study report.
- Temperature, humidity, in air chamber: The temperature inside the exposure chamber were measured by an electronic thermometer to : 20 to 24 °C. Chamber relative humidity was not measured due to the liquid nature of the test item.

TEST ATMOSPHERE
- Brief description of analytical method used: The test atmosphere was sampled nine times during the exposure period. A measured volume of air was drawn at a rate of 2 L/minute from an unused exposure port on the exposure chamber through a glass microfiber filter, mounted in an open face filter holder, using a wet type gas meter. Filters were weighed before and after sampling. The weight collected and the volume of air sampled were used to calculate the aerosol concentration. 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 eight 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:

no

Remarks:

nonspecific methods such as gravimetric filter analysis - utilised only

Duration of exposure:

4 h

Remarks on duration:

In accordance with the OECD TG 403 guidelines.

Concentrations:

Following an appropriate equilibration period, a single group 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 to be used for the first exposure. With further concentrations were selected after consideration of the results of the previous exposure. However, during preliminary characterisation trials a 5 mg/L test item aerosol atmosphere could not be achieved. A maximum practical exposure level was targeted. The mean achieved aerosol concentration value was lower than anticipated due to generation problems (atomizer needle blocking). 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
- 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 and two hours after termination of exposure and subsequently twice 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, 2, 4, 8 and 15 or after mortality.
- Necropsy of survivors performed: yes (and in the event of any mortalities) for survivors on day 14.
- 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, where appropriate or applicable. LC50 values and 95% confidence limits were calculated for males and females separately, where applicable.

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 2.08 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: maximum attainable concentration

Remarks:

During preliminary characterisation trials a 5 mg/L test item aerosol atmosphere could not be achieved. A maximum practical exposure level was targeted of 5 mg/L which yielded a 2.08 mg/L time-weighted mean achieved concentration

Mortality:

No mortalities during the study.

Clinical signs:

other: See "Other findings" for further information.

Body weight:

In group 1: 5.0 mg/L (target) / 2.08 mg/L mean achieved concentration : On day 2, following exposure slight body weight loss was observed in all males. Body weight gain was seen in four of five females with one female maintaining body weight on day 2. Body weight gain was observed on the next weighing occasion (day 4) for all males and two females. On Day 8, body weight gain was observed in all males/females and continued for the remainder of the observation period.

Gross pathology:

In group 1: 5.0 mg/L (target) / 2.08 mg/L mean achieved concentration : No macroscopic abnormalities were observed at the end of the observation period.

Other findings:

- Other observations: The respiratory tract was subjected to macroscopic examination for signs of irritancy or local toxicity during necropsy.
- Clinical signs: In group 1: 5.0 mg/L (target) / 2.08 mg/L mean achieved concentration : Immediately after the exposure, swaying was observed in two males and all females, unsteady gait was observed in all males/females. Swaying had resolved 1 hour post exposure. Unsteady gait had resolved in all males/females, with the exception of one male 2 hours post exposure. This sign had resolved by the end of day 1. Partially closed right eyelid was observed in one male immediately after exposure and chin
rubbing was observed in one male immediately after exposure. These signs had resolved in both males 1 hour post exposure. Wet rales were observed in one male and one female 1 hour post exposure. At 2 hours post exposure, wet rales were observed in one male and two females and were still present at the end of day 1 in all males/females observed with this sign. On the day following exposure, wet rales were present at the morning check in one male and one female. This sign had resolved by the end of day 2 and was not observed for the remaining observation period. Piloerection was observed for one male and one female 1 hour and 2 hours post exposure. At the end of day 1 piloerection was observed for these males/females and an additional female. This sign had resolved by the morning check of day 2. Dull eyes and vocalisation were observed for one female 1 hour post exposure and both signs had resolved by day 2. On one occasion, on Day 4, dry rales were observed at the morning check for one male. This had resolved by the end of day 4. There were no clinical signs at the end of the observation period.

Any other information on results incl. tables

Table 1.0 : Characteristics of the achieved atmosphere

Group Number	Target Concentration (mg/L)	Time-weighted mean achieved concentration (mg/L)	Mean Mass Median Aerodynamic Diameter (µm)	Geometric Standard Deviation	Comments
1	5.0	2.08	2.3	2.6	n=9 samples ; during preliminary characterisation trials a 5 mg/L test item aerosol atmosphere could not be achieved. A maximum practical exposure level was targeted. The mean achieved aerosol concentration value was lower than anticipated due to generation problems (atomizer needle blocking) ; TWA mean = 2.08 mg/L SD: 0.584

 

Table 2.0 : Mortality data

Group Number	Target Concentration (mg/L)	Time-weighted mean achieved concentration (mg/L)	Mortalities
			Female	Male
1	5.0	2.08	0/5	0/5

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the mean maximum attainable atmosphere concentration over 4 hours was 2.08 mg/L. This was the time-weighted mean exposure concentration achieved using a target concentration of 5.0 mg/L aerosol. The inhalation 4h-LC50 (male/female) was considered to be > 2.08 mg/L within the Wistar (RCCHan: WIST) strain rat.

Executive summary:

The study was performed according to OECD TG 403 accordance with GLP to assess the acute inhalation toxicity of the test item. A single group of ten RccHan : 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. During preliminary characterisation trials a 5 mg/L test item aerosol atmosphere could not be achieved. A maximum practical exposure level was targeted. The mean achieved aerosol concentration value was lower than anticipated due to generation problems (atomizer needle blocking). The characteristics of the atmosphere were as follows: Group 1: 5.00 mg/L (target concentration) or 2.08 mg/L time-weighted mean measured concentration. The characteristics of the achieved atmosphere was Mean Mass Median Diameter (particle size) and Inhalable Fraction <7 μm and the Geometric Standard Deviation was : 2.3 μm and 87%, was 2.6, respectively. There was no mortalities during the study. Immediately after the exposure, swaying was observed in two males and all females, unsteady gait was observed in all males/females. Swaying had resolved 1 hour post exposure. Unsteady gait had resolved in all males/females, with the exception of one male 2 hours post exposure. This sign had resolved by the end of day 1. Partially closed right eyelid was observed in one male immediately after exposure and chin rubbing was observed in one male immediately after exposure. These signs had resolved in both males 1 hour post exposure. Wet rales were observed in one male and one female 1 hour post exposure. At 2 hours post exposure, wet rales were observed in one male and two females and were still present at the end of day 1 in all males/females observed with this sign. On the day following exposure, wet rales were present at the morning check in one male and one female. This sign had resolved by the end of day 2 and was not observed for the remaining observation period. Piloerection was observed for one male and one female 1 hour and 2 hours post exposure. At the end of day 1 piloerection was observed for these males/females and an additional female. This sign had resolved by the morning check of day 2. Dull eyes and vocalisation were observed for one female 1 hour post exposure and both signs had resolved by day 2. On one occasion, on Day 4, dry rales were observed at the morning check for one male. This had resolved by the end of day 4. There were no clinical signs at the end of the observation period. On day 2, following exposure slight body weight loss was observed in all males. Body weight gain was seen in four of five females with one female maintaining body weight on day 2. Body weight gain was observed on the next weighing occasion (day 4) for all males and two females. On Day 8, body weight gain was observed in all males/females and continued for the remainder of the observation period. No macroscopic abnormalities were observed at the end of the observation period. Under the conditions of this study, the mean maximum attainable atmosphere concentration over 4 hours was 2.08 mg/L. The inhalation 4h-LC50 (male/female) was considered to be > 2.08 mg/L within the Wistar (RCCHan: WIST) strain rat.