Registration Dossier

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Administrative data

Description of key information

Oral dermal and inhalation toxicity

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:
26 September 2013 - 22 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in accordance with current OECD guidelines.
Qualifier:
according to guideline
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Deviations:
yes
Remarks:
See "Principles of method if other than guideline" for details of deviations.
Principles of method if other than guideline:
Deviation No.1 (12 November 2013) Due to a technician error, the Day 10 observation was not performed on the animal treated with the test item at a dose level of 300 mg/kg. This deviation was considered not to affect the purpose or integrity of the study.
GLP compliance:
yes (incl. QA statement)
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
Female Wistar (RccHan™:WIST) strain rats were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non-pregnant. After an acclimatization period of at least five days the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a number written on a cage card. At the start of the study the animals were eight to twelve weeks of age. The body weight variation did not exceed ±20% of the body weight of the initially dosed animal.The animals were housed in groups of up to four in suspended solid-floor polypropylene cages furnished with woodflakes. With the exception of an overnight fast immediately before dosing and for approximately three to four hours after dosing, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study.The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.Justification: Rats are the preferred species of choice as historically used for safety evaluation studies and are specified in the appropriate test guidelines.
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Remarks:
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.
Details on oral exposure:
For the purpose of the 2000 mg/kg dose level the test item was used as supplied. 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.The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted body weight at the time of dosing.
Doses:
300 ; 2000 mg/kg
No. of animals per sex per dose:
Preliminary test:300 mg/kg: 1 2000 mg/kg: 1Main test:2000 mg/kg: 1
Control animals:
no
Details on study design:
ProcedureIn the absence of data regarding the toxicity of the test item, 300 mg/kg was chosen as the starting dose.A single animal was treated with a dose level of 300 mg/ml (concentration of 30 mg/ml; dose volume 10 ml/kg). In the absence of toxicity at a dose level of 300 mg/kg, an additional animal was treated with a dose level of 300 mg/ml (specific gravity 0.813; dose volume 2.47 ml/kg). In the absence of toxicity at a dose level of 2000 mg/kg, an additional group of 4 animals was treated with a dose level of 300 mg/ml (specific gravity 0.813; dose volume 2.47 ml/kg).A total of five animals were therefore treated at a dose level of 2000 mg/kg in the study.All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted body weight at the time of dosing. Treatment of animals was sequential. Sufficient time was allowed between each dose level to confirm the survival of the previously dosed animals.Clinical observations were made ½, 1, 2, and 4 hours after dosing and then daily for fourteen days. Morbidity and mortality checks were made twice daily. Due to a technician error, the Day 10 observation was not performed on the animal treated with the test item at a dose level of300 mg/kg. This deviation was considered not to affect the purpose or integrity of the study. Individual body weights were recorded on Day 0 (the day of dosing) and on Days 7 and 14.At the end of the observation period the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.Evaluation of dataThe test item will be classified according to Annex 3 of the OECD Guidelines for Testing of Chemicals No. 420 "Acute Oral Toxicity - Fixed Dose Method" (adopted 17 December 2001).Evaluation of data included identification of the number of animals that died during the study (or that were killed for humane reasons), and determination of the nature, severity, onset and duration of the toxic effects. If possible, the signs of evident toxicity were described. Evident toxicity refers to the toxic effects of sufficient severity that administration of the next higher dose level could result in development of severe signs of toxicity and probable mortality. Effects on body weights and abnormalities noted at necropsy were also identified.Using the mortality data obtained, an estimate of the acute oral median lethal dose (LD50) of the test item was made.
Statistics:
None
Preliminary study:
No effects were noted in either the 300 or 2000 mg/kg prelimary dosing studies.
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: No effects noted at highest dose level
Mortality:
There was no mortality.
Clinical signs:
No signs of systemic toxicity were noted during the observation period.
Body weight:
All animals showed expected gains in body weight over the observation period.
Gross pathology:
No abnormalities were noted at necropsy.

 Individual Clinical Observations and Mortality Data -2000mg/kg

Dose Level mg/kg

Animal Number and Sex

Effects Noted After Dosing
(Hours)

Effects Noted During Period After Dosing
(Days)

½

1

2

4

1

2

3

4

5

6

7

8

9

10

11

12

13

14

2000

2-0

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-0

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-1

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-2

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-3

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0


0=   No signs of systemic toxicity

Interpretation of results:
not classified
Remarks:
Migrated informationCriteria used for interpretation of results: EU
Conclusions:
The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg body weight. No classification is applicable.
Executive summary:

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg body weight. No classification is applicable.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
1

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:
6 July 2016 to 26 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study was conducted in accordance with OECD and EU test guidelines and conducted in an accredited GLP laboratory
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
yes
Remarks:
see "Principles of method if other than guideline" for more details.
Qualifier:
according to guideline
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
yes
Remarks:
see "Principles of method if other than guideline" for more details.
Principles of method if other than guideline:
Deviations:Deviation No 1 After an acclimatization period of at least 5 days the animals were given a number unique within the study by ear punching. Deviation No 2 The Archiving statement has been amended to reflect the fact that no Specimens were retained during this study. Therefore, the following sentence has been removed: Specimens that no longer afford evaluation will be discarded in accordance with Standard Operating Procedures and without further notice. These deviations were considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Test type:
traditional method
Limit test:
no
Specific details on test material used for the study:
Identification: NovaSpec Base OilPhysical state/appearance: Clear colorless, slightly viscous liquidBatch: TS17605Purity: 100 %Expiry date: 01 June 2018Storage conditions: Room temperature, in the dark
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal InformationMale and female RccHan™ : WIST strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were randomly allocated to cages. After an acclimatization period of at least 5 days the animals were given a number unique within the study by ear punching and a number written on a color coded cage card. At the start of the study the animals were approximately 8 to 12 weeks old and within the weight range of 200 g to 350 g. The females were nulliparous and non pregnant.Animal Care and HusbandryThe animals were housed in groups of up to five by sex in solid floor polypropylene cages with stainless steel lids, furnished with softwood flakes. With the exception of the exposure period, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study.The temperature and relative humidity were set to achieve limits of 19 to 25° C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness. The animals were retained in this accommodation at all times except during the exposure period.The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Route of administration:
inhalation: mist
Type of inhalation exposure:
nose only
Vehicle:
not specified
Mass median aerodynamic diameter (MMAD):
1.37 µm
Geometric standard deviation (GSD):
3
Remark on MMAD/GSD:
Inhalable Fraction (% <4 µm) = 83.7Mean Achieved Atmosphere Concentration (mg/L) =5.09
Details on inhalation exposure:
Atmosphere GenerationThe test item was aerosolized using a metal concentric jet nebulizer (Envigo CRS Limited, UK) 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.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.The cylindrical exposure chamber had a volume of approximately 30 liters (dimensions: 28 cm diameter x 50 cm high). The concentration within the exposure chamber was controlled by adjusting the rate of the infusion pump. 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 diagram of the dynamic (continuous flow) system employed is shown in Figure 1.Homogeneity of the test atmosphere within the chamber was not specifically determined during this study. Chambers of the same design (ADG Developments Ltd, Hitchin, Herts, UK) have been fully validated and shown to produce evenly distributed atmospheres in the animals’ breathing zone with a wide variety of test items (Green J D et al, 1984).Prior to the start of the study, test item atmospheres were generated within the exposure chamber. During this characterization period test item input rates were varied in an attempt to achieve the required atmospheric conditions.Exposure ProcedureOne day 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.Following an appropriate equilibration period a single group of ten rats (five males and five females) was exposed to an atmosphere of the test item for a period of 4 hours. A target concentration of 5.0 mg/L was used for the exposure. As the mean achieved concentration was 102 % of target and no deaths occurred, no further levels were required. Exposure Chamber Temperature and Relative HumidityThe temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter (Hanna Instruments Ltd, Beds., UK) located in a vacant port in the animals’ breathing zone of the chamber and recorded every 30 minutes throughout the 4 Hour exposure period. Exposure Chamber Oxygen ConcentrationOxygen levels within the exposure chamber were measured by an electronic oxygen analyzer (Servomex (UK) Ltd, Crowborough, East Sussex) located in a port in the animals breathing zone during the 4 Hour exposure period. The test atmosphere was generated to contain at least 19% oxygen. Exposure Chamber Atmosphere ConcentrationPrior to the inhalation phase of the study, the non-volatile component of the test item was determined by adding a small, known amount of test item to glass fiber filters and recording their weights. The filters were then dried in a desiccator at room temperature for approximately 24 hours and then weighed again. The difference in the two weights was taken as the volatile content of the test item and the non-volatile component was calculated as a percentage. The mean non-volatile component of the batch used during the formal exposure was considered to be 100 % (n=10). As the test item did not show signs of containing a volatile component it was considered acceptable to determine test atmosphere concentrations within the test chamber by using wet filter weights only.The actual chamber concentration was measured at regular intervals during the exposure period. The gravimetric method used glass fiber filters placed in a filter holder. The holder was temporarily sealed in a vacant port in the exposure chamber in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through the filter using a vacuum pump.Each filter was weighed before and after sampling in order to calculate the weight of collected test item. The difference in the two weights, divided by the volume of atmosphere sampled, gave the actual chamber concentration.The nominal chamber concentration was calculated by dividing the mass of test item disseminated into the chamber by the total volume of air that flowed through the chamber during the exposure.The nominal concentration was 283 % of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was relatively straight forward.
Analytical verification of test atmosphere concentrations:
no
Duration of exposure:
4 h
Concentrations:
The test atmosphere was sampled seventeen times during the exposure period and the actual concentration of the test item calculated. Mean atmospheric concentration 5.09 mg/L
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
not specified
Details on study design:
Observations
Clinical Signs
All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, 1 hour after termination of exposure and subsequently once daily for 14 days. Any evidence of overt toxicity was recorded at each observation.
Body Weight
Individual body weights were recorded on arrival, prior to treatment on the day of exposure (Day 0) and on Days 1, 3, 7 and 14.

Terminal Investigations
Necropsy
At the end of the 14 day observation period the animals were killed by intravenous overdose of sodium pentobarbitone. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.

Particle Size Distribution
The particle size of the generated atmosphere inside the exposure chamber was determined three times during the exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK). This device consisted of six impactor stages (8.4, 7.3, 3.6, 1.3, 0.94 and 0.43 µm cut points) with stainless steel collection substrates and a backup glass fiber filter, housed in an aluminum sampler. The sampler was temporarily sealed in a sampling port in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through it using a vacuum pump.The collection substrates and backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by difference.The mean amount for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than 8.4, 7.3, 3.6, 1.3, 0.94 and 0.43 µm was calculated.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.
Statistics:
Data EvaluationData evaluations included the relationship, if any, between the animals’ exposure to the test item and the incidence and severity of all abnormalities including behavioral and clinical observations, necropsy findings, body weight changes, mortality and any other toxicological effects.Using the mortality data obtained, an estimate of the acute inhalation median lethal concentration (LC50) of the test item was made.
Preliminary study:
Not applicable
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.09 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No mortality occured
Clinical signs:
other: Signs of hunched posture and pilo-erection are commonly seen in animals for short periods on removal from the chamber following 4-Hour inhalation studies. Wet fur is commonly recorded both during and for a short period after exposure. These observations a
Body weight:
All male animals and three females exhibited body weight losses on the first day post-exposure. Body weight gains were noted for all male animals during the remainder of the recovery period. In contrast, four female animals exhibited body weight losses or showed no body weight gain from Days 3 to 7 post-exposure. All female animals exhibited body weight gains during the final week of the recovery period.
Gross pathology:
No macroscopic abnormalities were detected amongst animals at necropsy.
Other findings:
No other information available.

Individual Clinical Observations – (Day of Exposure)

Mean Achieved Atmosphere Concentration (mg/L)

Animal Number and Sex

Effects Noted During Exposure
(Hours)

Effects Noted on Removal from Chamber

Effects Noted 1 Hour Post Exposure

1

2

3

5.09

1 Male

WfRd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

2 Male

Wf Rd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

3 Male

Wf Rd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

4 Male

Wf Rd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

5 Male

Wf Rd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

6 Female

Wf Rd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

7 Female

Wf Rd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

8 Female

Wf Rd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

9 Female

Wf Rd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

10 Female

Wf Rd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

 Key:

Wf= Wet Fur         Rd = Decreased respiratory rate      H = Hunched posture        P = Pilo-erection        Ri = Increased respiratory rate        

Individual Clinical Observations – Recovery Period

Mean Achieved Atmosphere Concentration (mg/L)

Animal Number and Sex

Effects Noted Post Exposure
(Days)

1

2

3

4

5

6

7

8-14

5.09

1 Male

H

0

0

0

0

0

0

0

2 Male

H

0

0

0

0

0

0

0

3 Male

H

0

0

0

0

0

0

0

4 Male

H

0

0

0

0

0

0

0

5 Male

H

0

0

0

0

0

0

0

6 Female

H

0

0

0

0

0

0

0

7 Female

H

0

0

0

0

0

0

0

8 Female

H

0

0

0

0

0

0

0

9 Female

H

0

0

0

0

0

0

0

10 Female

H

0

0

0

0

0

0

0

 Key:

H= Hunched posture         0 = No abnormalities detected

Individual Body Weights and Body Weight Changes

Mean Achieved Atmosphere Concentration (mg/L)

Animal Number and Sex

Body Weight (g) at Day

Body Weight Change (g) During Days

-6

0

1

3

7

14

-6 to 0

0 to 1

1 to 3

3 to 7

7 to 14

5.09

1 Male

200

240

239

248

271

299

40

-1

9

23

28

2 Male

200

228

226

233

246

259

28

-2

7

13

13

3 Male

208

240

239

249

267

281

32

-1

10

18

14

4 Male

202

237

236

245

268

290

35

-1

9

23

22

5 Male

210

243

242

250

270

291

33

-1

8

20

21

6 Female

203

215

214

215

215

219

12

-1

1

0

4

7 Female

201

206

205

209

205

207

5

-1

4

-4

2

8 Female

200

209

201

206

213

220

9

-8

5

7

7

9 Female

200

212

213

215

211

216

12

1

2

-4

5

10 Female

196

204

205

207

205

208

8

1

2

-2

3

 

 Individual Necropsy Findings

Mean Achieved Atmosphere Concentration (mg/L)

Animal Number
and Sex

Time of Death

Macroscopic Observations

5.09

1 Male

Terminal Kill Day 14

No abnormalities detected

2 Male

Terminal Kill Day 14

No abnormalities detected

3 Male

Terminal Kill Day 14

No abnormalities detected

4 Male

Terminal Kill Day 14

No abnormalities detected

5 Male

Terminal Kill Day 14

No abnormalities detected

6 Female

Terminal Kill Day 14

No abnormalities detected

7 Female

Terminal Kill Day 14

No abnormalities detected

8 Female

Terminal Kill Day 14

No abnormalities detected

9 Female

Terminal Kill Day 14

No abnormalities detected

10 Female

Terminal Kill Day 14

No abnormalities detected

 

Interpretation of results:
GHS criteria not met
Conclusions:
No deaths occurred in a group of ten rats exposed to a mean achieved atmosphere concentration of 5.09 mg/L for 4 hours. It was therefore considered that the acute inhalation median lethal concentration (4 hour LC50) of the test item in the Wistar strain rat was greater than 5.09 mg/L and as such is not classified for Acute Inhalation Toxicity.
Executive summary:

A study was performed to assess the acute inhalation toxicity of the test item.The method used was designed to be compatible with that described in the OECD Guideline for Testing of Chemicals No. 403 “Acute Inhalation Toxicity” (2009) and Method B.2. (Inhalation) of Commission Regulation (EC) No. 440/2008.

A group of ten RccHanTM: WIST strain rats (five males and five females) was exposed to an aerosol atmosphere. The animals were exposed for 4 hours using a nose only exposure system, followed by a fourteen day observation period.

Atmospheric conditions were as follows:

Mean Achieved Atmosphere Concentration (mg/L) 5.09

Mean Mass Median Aerodynamic Diameter (µm) 1.37

Inhalable Fraction (% <4 µm) 83.7

Geometric Standard Deviation 3.00

Clinical Observations: Common abnormalities noted during the study included decreased respiratory rate, increased respiratory rate, hunched posture, pilo-erection and wet fur. Animals recovered to appear normal on Day 2 post-exposure.

Body Weight: All male animals and three females exhibited body weight losses on the first day post-exposure. Body weight gains were noted for all male animals during the remainder of the recovery period.  In contrast, four female animals exhibited body weight losses or showed no body weight gain from Days 3 to 7 post-exposure. All female animals exhibited body weight gains during the final week of the recovery period.

 

Necropsy: No macroscopic abnormalities were detected amongst animals at necropsy.

 

In conclusion acute inhalation median lethal concentration (4 hour LC50) of the test item in the Wistar strain rat was greater than 5.09 mg/L and as such does not meet the criteria to classify for Acute Inhalation Toxicity.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LC50
Value:
5.09 mg/m³
Quality of whole database:
1

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Type of information:
other: literature assessment of topical exposure
Adequacy of study:
key study
Study period:
17 August 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The Klimish rating is considered appropriate in accordance with ANNEX XI -GENERAL RULES FOR ADAPTATION OF THE STANDARD TESTING REGIME SET OUT IN ANNEXES VII TO X, Para 1.2 which states that: 1.2. Weight of evidence There may be sufficient weight of evidence from several independent sources of information leading to the assumption/conclusion that a substance has or has not a particular dangerous property, while the information from each single source alone is regarded insufficient to support this notion. There may be sufficient weight of evidence from the use of newly developed test methods, not yet included in the test methods referred to in Article 13(3) or from an international test method recognised by the Commission or the Agency as being equivalent, leading to the conclusion that a substance has or has not a particular dangerous property. Where sufficient weight of evidence for the presence or absence of a particular dangerous property is available: – further testing on vertebrate animals for that property shall be omitted, – further testing not involving vertebrate animals may be omitted. In all cases adequate and reliable documentation shall be provided. The literature paper presented presents evidence from a number of studies conducted using mineral hydrocarbon oil and exposure conducted topically. In each study, evidence is presented for the lack of absorption topically, as well as no evidence of irritation or toxicity noted. This, in conjunction with the in vitro studies indicates that there is low potential for absorption. In accordance with REACH Annex VIII, section 8.5.3, this study should be conducted in the event that the physicochemical and toxicological properties suggest potential for a significant rate of absorption through the skin. This is not the case for a substance of this type.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Various exposure durations and applications are cited within the literature paper. These are summarised below, and the literature paper is attached for reference purposes.
GLP compliance:
not specified
Test type:
other: weight of evidence on topical applications.
Limit test:
yes
Species:
other: mice, rats and rabbits
Strain:
not specified
Sex:
male/female
Type of coverage:
other: various
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
Several lifetime, topical exposure studies of petroleum distillates and oils, using white mineral oil as a negative control have been conducted (Biles et al., 1988; McKee et al., 1986, 1989 and 1990; McKee and Lewis, 1987). A summary of the design and results of these studies is presented in the table below. In general, male C3H mice were exposed topically on the shaved dorsal surface, two to three times each week for at least 24 months, to a variety of petroleum distillates, oils or Mineral Oil, USP. Importantly, in each study, a complete autopsy was performed and histological evaluation included the liver, kidney, spleen and mesenteric lymph nodes. The average dose of mineral oil delivered topically to the mice was 296 mg/kg/day, depending on the density of the material. There was no evidence of any treatment-related histopathological changes in any of the organs examined after lifetime topical exposure to white Mineral Oil, USP. In addition, there was no evidence of turnout formation either at the site of application or in any of the internal organs examined in mice treated with white mineral oil. Topical exposure to white mineral oils in this species caused no measurable detrimental effects based on these data.Topical 90-day studies using refined mineral oil as a negative control have been conducted by the National Toxicology Program (NTP) (1992) (see below). Male and female F344/N rats and C3H mice were exposed topically to mineral oil, approximately 41 mg/kg/day and 143 mg/kg/day, respectively, for 91 days. Complete autopsy and histopathological examination were performed on each animal. In addition, selected organ weights were obtained. Finally, the results obtained in topical administration studies using rabbits are presented in the table below (Johnson & Johnson Consumer Products, 1992, unpublished data). New Zealand White rabbits were exposed topically to products containing either 16 or 99% mineral oil. In both studies, the rabbits were administered 2 g/kg/day of finished product over 10% of the body surface area for 20 days. Test material was not administered for 14 days before sacrifice. A complete autopsy and histopathological examination was performed on each animal.
Duration of exposure:
Variable. See tabulated data below.
Doses:
Variable. See tabulated data below.
No. of animals per sex per dose:
Variable. See tabulated data below.
Control animals:
not specified
Details on study design:
Variable. See tabulated data below and details listed above under "Details on Dermal Exposure".
Statistics:
None reported.
Sex:
male
Dose descriptor:
other: lifetime exposure
Effect level:
> 296 other: mg/kg/day
Based on:
test mat.
Remarks on result:
other: 4 separate studies on C3H/HeJ mice at this dose level are reported. No effects or toxicity is noted in any study
Sex:
male
Dose descriptor:
other: lifetime exposure
Effect level:
> 238 other: mg/kg/day
Based on:
test mat.
Remarks on result:
other: Study on C3H/HeJ mice at this dose level are reported. No effects or toxicity are noted.
Sex:
male/female
Dose descriptor:
other: 91 day exposure
Effect level:
> 41 other: mg/kg/day
Based on:
test mat.
Remarks on result:
other: Study on F344 Rat at this dose level are reported. Histopathological observations in the skin or internal organs of either species exposed to mineral oil were within normal limits.
Sex:
male/female
Dose descriptor:
other: 91-day exposure
Effect level:
> 143 other: mg/kg/day
Based on:
test mat.
Remarks on result:
other: Study on C3H mice at this dose level are reported. Histopathological observations in the skin or internal organs of either species exposed to mineral oil were within normal limits.
Sex:
male/female
Dose descriptor:
other: 21 day exposure
Effect level:
> 2 000 other: mg/kg/day
Based on:
test mat.
Remarks on result:
other: Study on New Zealand White Rabbit at this dose level are reported; 99% and 16% solutions. There was no evidence of dermal, haematological or histopathological changes associated with administration of products containing mineral oil.
Mortality:
No data reported; assumed that no changes were noted due to the lack of effects observed.
Clinical signs:
No data reported; assumed that no changes were noted due to the lack of effects observed.
Body weight:
No data reported; assumed that no changes were noted due to the lack of effects observed.
Gross pathology:
An increase in liver and kidney weights was observed in the male and female F344/N rats treated topically with mineral oil; liver weights were increased in both sexes of C3H mice. Histopathological observations in the skin or internal organs of either species exposed to mineral oil were within normal limits. The NTP concluded that only cutaneous irritation in the mouse represented treatment-related toxicity following topical exposure to Mineral Oil, USP. This effect is surprising in that previous studies involving topical exposure to mineral oil have not resulted in irritation and this finding is inconsistent with the emollient properties (i.e. prevention of water loss) of mineral oil (Blank, 1988).
Other findings:
Conclusions on dermal absorption are based on the results of experiments with hydrocarbon surrogates. Rossmiller and Hoekstra (1966), reported that 48 hr after the application of [14C] hexadecane in mineral oil to the backs of guinea pigs only a very small amount, less than 0.1% of the applied dose, was found in the dermis. Following a single topical application (2.0 g/kg) of chlorinated paraffins to rats, Yang et al. (1987) reported that less than 1% of C18 paraffin and less than 0.1% of C28 paraffin was recovered in faeces, urine, expired air or tissues over 96 hr. In vitro human skin penetration studies with [14C] n-pentadecane and [14C] n-undecane in chlorinated paraffins have reported 0% and less than 0.01% of the applied dose, respectively, in the receptor fluid after 54 hr (Scott, 1989). The results of microscopic studies suggest that the depth of penetration of hydrocarbons such as octadecane is limited to the stratum corneum in mouse (Ghadially et al., 1992) and human skin (Zesch and Bauer, 1985). Collectively, these data support the view that mineral oil does not effectively penetrate the skin beyond the stratum corneum, resulting in minimal (<1%) absorption of white mineral oils after topical exposure.More recently, the results of studies by Brown et al. (1995), Diembeck and Duesing (1993) and Diembeck and Grimmert (1993) investigating percutaneous absorption of [14C] hexadecane and [3H] docosane from petrolatum or mineral oil after topical application to excised pig skin have confirmed and extended earlier studies. The distribution of the test material between the skin surface, horny layer, epidermis, dermis and receptor fluid was determined. Percutaneous absorption and dermal penetration of [14C] hexadecane and [3H] docosane from petrolatum and Mineral Oil, USP was measured 24 hr after topical application to excised pig skin. Neither radiolabelled compound was measured in the receptor fluid and, in the dermis, 0.8% or less of the total radioactivity was measured 24 hr after application to the skin. These data are further evidence that topically applied mineral hydrocarbons penetrate the skin barrier very slowly and that less than 1% reaches the dermis.On the basis of these findings and reports of negligible epidermal penetration of topically applied white mineral oils, there is no evidence of any hazard identified for topical exposure to white mineral oils at any dose in multiple species, and only minor deposition may occur in the stratum corneum.. This conclusion is supported by the long and uneventful human use of white mineral oils in drug and non-drug topically applied products.
Interpretation of results:
not classified
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
All available evidence suggests that subchronic or chronic topical exposure to highly refined white mineral oils does not produce gross or histopathological changes in the internal organs or at the site of application in either C3H mice, F344/N rats or New Zealand White rabbits. Similarly, there is no evidence that chronic topical exposure to white mineral oils produces any adverse effects or shortens the lifespan of animals. On the basis of this review of the available literature, it seems unlikely that sufficient amounts of mineral hydrocarbons will be absorbed after topical administration to result in systemic concentrations high enough to cause deposition in the mesenteric lymph nodes and/or liver and thus lead to histiocytosis and granuloma formation. In this regard, following subchronic and chronic topical application there have been no reported effects on any internal organ system attributable to white mineral oils. Considering the long history of use of white mineral oils in topical applied products, the lack of any substantive toxicological findings in rodents and humans suggests the absence of any health risk.
Executive summary:

A full justification for the read across applied for this substance is contained within Section 1.4, reference " Explanation of NovaSpec Base Oil - FINAL". This document contains a full explanation of the manufacturing route for the substance and discussion on why this substance should be considered as a UVCB white oil. Physical properties of the crude reaction product and the different product grades (or distillate fractions) derive from the general chemical structure characteristics (linear-branched alkanes with characteristic branching length and position). Specific unique chemical structures are not isolated in any process step and due to the tens of thousands of isomers and the high degree and variable nature branching and chain length, the base oil bulk properties are the result of the average structure characteristics. The unique viscosity grades each contain all the same typical chemical structures and predominantly overlapping Molecular Weight distributions. The unique viscosity and volatility characteristics of each viscosity grade derive from the boiling point distribution, a result of the short-path distillation (wiped film evaporation). This is identical to the production of petroleum derived white oils.

In order to avoid the requirement to further test a substance that is a derived white oil on animals, a weight of evidence approach is utilised to provide evidence of lack of dermal toxicity. This is via the utilisation of various literature papers.

Conclusions on dermal absorption are based on the results of experiments with hydrocarbon surrogates. Rossmiller and Hoekstra (1966), reported that 48 hr after the application of [14C] hexadecane in mineral oil to the backs of guinea pigs only a very small amount, less than 0.1% of the applied dose, was found in the dermis. Following a single topical application (2.0 g/kg) of chlorinated paraffins to rats, Yang et al. (1987) reported that less than 1% of C18 paraffin and less than 0.1% of C28 paraffin was recovered in faeces, urine, expired air or tissues over 96 hr. In vitro human skin penetration studies with [14C] n-pentadecane and [14C] n-undecane in chlorinated paraffins have reported 0% and less than 0.01% of the applied dose, respectively, in the receptor fluid after 54 hr (Scott, 1989). The results of microscopic studies suggest that the depth of penetration of hydrocarbons such as octadecane is limited to the stratum corneum in mouse (Ghadially et al., 1992) and human skin (Zesch and Bauer, 1985). Collectively, these data support the view that mineral oil does not effectively penetrate the skin beyond the stratum corneum, resulting in minimal (<1%) absorption of white mineral oils after topical exposure.

More recently, the results of studies by Brown et al. (1995), Diembeck and Duesing (1993) and Diembeck and Grimmert (1993) investigating percutaneous absorption of [14C] hexadecane and [3H] docosane from petrolatum or mineral oil after topical application to excised pig skin have confirmed and extended earlier studies. The distribution of the test material between the skin surface, horny layer, epidermis, dermis and receptor fluid was determined. Percutaneous absorption and dermal penetration of [14C] hexadecane and [3H] docosane from petrolatum and Mineral Oil, USP was measured 24 hr after topical application to excised pig skin. Neither radiolabelled compound was measured in the receptor fluid and, in the dermis, 0.8% or less of the total radioactivity was measured 24 hr after application to the skin. These data are further evidence that topically applied mineral hydrocarbons penetrate the skin barrier very slowly and that less than 1% reaches the dermis.

On the basis of these findings and reports of negligible epidermal penetration of topically applied white mineral oils, there is no evidence of any hazard identified for topical exposure to white mineral oils at any dose in multiple species, and only minor deposition may occur in the stratum corneum.. This conclusion is supported by the long and uneventful human use of white mineral oils in drug and non-drug topically applied products.

All available evidence suggests that subchronic or chronic topical exposure to highly refined white mineral oils does not produce gross or histopathological changes in the internal organs or at the site of application in either C3H mice, F344/N rats or New Zealand White rabbits. Similarly, there is no evidence that chronic topical exposure to white mineral oils produces any adverse effects or shortens the lifespan of animals. On the basis of this review of the available literature, it seems unlikely that sufficient amounts of mineral hydrocarbons will be absorbed after topical administration to result in systemic concentrations high enough to cause deposition in the mesenteric lymph nodes and/or liver and thus lead to histiocytosis and granuloma formation. In this regard, following subchronic and chronic topical application there have been no reported effects on any internal organ system attributable to white mineral oils. Considering the long history of use of white mineral oils in topical applied products, the lack of any substantive toxicological findings in rodents and humans suggests the absence of any health risk.

A NOAEL was not reported, as no effects were noted but is proposed to be greater than or equal to 2000 mg/kg/day (the highest dose tested) based on the lack of treatment-related effects.

This study received a Klimisch score of two and is classified as reliable with restriction because it is an acceptable and well-documented literature study report following basic scientific principles.

No classification is applicable.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
2

Additional information

Acute Oral Toxicity

The substance is not considered to be harmful by oral exposure, with an LD50 > 2000 mg/kg in a study conducted in accordance with OECD / EU test guidelines in a GLP accredited laboratory.

 

Acute Dermal Toxicity

Conclusions on dermal absorption are based on the results of experiments with hydrocarbon surrogates. Rossmiller and Hoekstra (1966), reported that 48 hr after the application of [14C] hexadecane in mineral oil to the backs of guinea pigs only a very small amount, less than 0.1% of the applied dose, was found in the dermis. Following a single topical application (2.0 g/kg) of chlorinated paraffins to rats, Yang et al. (1987) reported that less than 1% of C18 paraffin and less than 0.1% of C28 paraffin was recovered in faeces, urine, expired air or tissues over 96 hr. In vitro human skin penetration studies with [14C] n-pentadecane and [14C] n-undecane in chlorinated paraffins have reported 0% and less than 0.01% of the applied dose, respectively, in the receptor fluid after 54 hr (Scott, 1989). The results of microscopic studies suggest that the depth of penetration of hydrocarbons such as octadecane is limited to the stratum corneum in mouse (Ghadially et al., 1992) and human skin (Zesch and Bauer, 1985). Collectively, these data support the view that mineral oil does not effectively penetrate the skin beyond the stratum corneum, resulting in minimal (<1%) absorption of white mineral oils after topical exposure.

More recently, the results of studies by Brown et al. (1995), Diembeck and Duesing (1993) and Diembeck and Grimmert (1993) investigating percutaneous absorption of [14C] hexadecane and [3H] docosane from petrolatum or mineral oil after topical application to excised pig skin have confirmed and extended earlier studies. The distribution of the test material between the skin surface, horny layer, epidermis, dermis and receptor fluid was determined. Percutaneous absorption and dermal penetration of [14C] hexadecane and [3H] docosane from petrolatum and Mineral Oil, USP was measured 24 hr after topical application to excised pig skin. Neither radiolabelled compound was measured in the receptor fluid and, in the dermis, 0.8% or less of the total radioactivity was measured 24 hr after application to the skin. These data are further evidence that topically applied mineral hydrocarbons penetrate the skin barrier very slowly and that less than 1% reaches the dermis.

 

On the basis of these findings and reports of negligible epidermal penetration of topically applied white mineral oils, there is no evidence of any hazard identified for topical exposure to white mineral oils at any dose in multiple species, and only minor deposition may occur in the stratum corneum.. This conclusion is supported by the long and uneventful human use of white mineral oils in drug and non-drug topically applied products.

Other animal studies with mineral oils suggest that absorption after inhalation exposure is slow and that lung clearance may be mediated by macrophage phagocytosis. Oil-filled macrophages have been found to accumulate in the lungs. Particle size and composition may influence the absorption of inhaled mineral oil components.

 

Acute Inhalation Toxicity

No deaths occurred in a group of ten rats exposed to a mean achieved atmosphere concentration of 5.09 mg/L for 4 hours. It was therefore considered that the acute inhalation median lethal concentration (4 hour LC50) of the test item in the Wistar strain rat was greater than 5.09 mg/L and as such is not classified for Acute Inhalation Toxicity.

 

Justification for selection of acute toxicity – oral endpoint 

Study was conducted in accordance with OECD / EU test guidelines in a GLP accredited laboratory.

Justification for selection of acute toxicity – dermal endpoint 

Suitable literature paper assessing a number of documented studies.

Justification for selection of acute toxicity - inhalation endpoint

Study was conducted in accordance with OECD / EU test guidelines in a GLP accredited laboratory.

Justification for classification or non-classification

Oral Toxicity

This study received a Klimisch score of one and is classified as reliable without restriction because it is an acceptable and well-documented study report conducted in accordance with GLP and to current guidelines. LD50 was determined to be > 2000 mg/kg.

Dermal Toxicity

This study received a Klimisch score of two and is classified as reliable with restriction because it is an acceptable and well-documented literature study report following basic scientific principles. LD50 was determined to be > 2000 mg/kg.

Inhalation Toxicity

This study received a Klimisch score of one and is classified as reliable without restriction because it is an acceptable and well-documented study report conducted in accordance with GLP and to current guidelines. LC50 was determined to be > 5.09 mg/L

No classification is applicable.

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