Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids

Endpoint summary

Description of key information

Oral: LD50 > 2000 mg/kg bw

Read-across from structural analogue source substances Dipentaerythritol hexaesters with fatty acids, C5 and C9iso (CAS No. 647028-25-9), Hexanoic acid, 2-ethyl-, 2,2-bis [ [(2-ethyl-1-oxohexyl)oxy] methyl] -1,3-propanediyl ester (CAS No. 7299-99-2), Pentaerythritol ester of pentanoic acids, mixed esters with pentaerythritol, isopentanoic and isononanoic acid (CAS No. 146289-36-3), Fatty acids, C5-9 tetraesters with pentaerythritol (CAS No. 67762-53-2), and Pentaerythritol tetraesters of n-decanoic, n-heptanoic, n-octanoic and n-valeric acids (CAS No. 68424-31-7)

Inhalation: LC50 > 5 mg/L air

Read-across from structural analogue source substances Fatty acids, C5-9 tetraesters with pentaerythritol (CAS No. 67762-53-2), Fatty acids, C5-9, mixed esters with dipentaerythritol and pentaerythritol (CAS No. 85536-35-2) and Pentaerythritol tetraesters of n-decanoic, n-heptanoic, n-octanoic and n-valeric acids (CAS No. 68424-31-7)

Dermal (OECD 402, GLP): > 2000 mg/kg bw

Read-across from structural analogue source substance Dipentaerythritol with fatty acids, C5 and C9iso (CAS No. 647028-25-9)

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08. Jan. - 29. Jan. 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 (Acute Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: Wistar TNO

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Weight at study initiation: mean 190 g (male), 160 g (female)
- Fasting period before study: 16 hours
- Housing: 5 animals per cage: Makrolon type-3 with standard soft wood bedding
- Diet: pelleted maintenance diet 1324 (Altromin GmbH), ad libitum
- Water: community tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25
- Humidity (%): 45-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: arachidis oil, DAB9

Details on oral exposure:

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: weighing one day before application, on day 2, 7 and 14 after application, animals were checked for mortality/viability two times daily
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, other: macroscopic examination of the cranial, thoracic and visceral cavities

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occurred during the study period.

Clinical signs:

other: No clinical signs of toxicity were observed up to the end of the 14-day observation period.

Gross pathology:

Necropsy and histopathological examination revealed no substance-related findings.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07 Dec - 23 Dec 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, UK

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

other: Sprague-Dawley CD (Crl:CD® (SD) IGS BR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Ltd, Margate, Kent, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 209 – 229 g (males) and 204 - 235 g (females)
- Fasting period before study: animals were fasted overnight prior to administration.
- Housing: animals were housed in groups of up to 3 per cage per sex in solid-floor polypropylene cages furnished with woodflakes.
- Diet: Rat and Mouse Expanded Diet No. 1 (Special Diets Services Limited, Witham, Essex, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Example: 19-21
- Humidity (%): 37-67
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED: 2.06 mL/kg bw

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: The information available suggested a starting dose of 2000 mg/kg bw.

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

3

Control animals:

other: not required

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: animals were observed 30 min, 1, 2 and 4 h after dosing and subsequently daily for 14 days and individual body weights were determined prior to dosing and 7 and 14 days after treatment.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical observations, body weight

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

5 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: LD50 cut-off according to OECD 423

Mortality:

No mortality occurred during the study period.

Clinical signs:

other: No clinical signs of toxicity were observed up to the end of the 14-day observation period.

Gross pathology:

Necropsy revealed no substance-related findings.

Table 1. Individual body weights and weekly body weight changes.

Dose level mg/kg bw	Animal number and sex	Body weight (g) at Day			Body weight gain (g) during week
		0	7	14	1	2
2000	1-0 Female	204	237	249	33	12
2000	1-1 Female	235	262	280	27	18
2000	1-2 Female	225	265	284	40	19
2000	1-0 Male	217	300	352	83	52
2000	1-1 Male	209	284	318	75	34
2000	1-2 Male	229	297	339	68	42

 

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Reference 3

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

7 Dec - 23 Dec 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)

Version / remarks:

adopted in 1992

Deviations:

yes

Remarks:

no analytical purity reported

GLP compliance:

no

Test type:

fixed dose procedure

Limit test:

yes

Species:

rat

Strain:

other: Albino Rats (Outbred). Stock: Sprague-Dawley-derived (CD) [Crl: CD (SD) IGS BR]

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Kingston, New York, USA
- Age at study initiation: 9-12 weeks
- Weight at study initiation: 279-318 g (males), 204-219 g (females)
- Fasting period before study: animals were fasted overnight prior administration
- Housing: 2 to 6 animals of the same sex per cage (during acclimation) und individual (during study) per cages. Cages were suspended, stainless cages with wire mesh bottoms.
- Diet: certified Rodent Diet, No. 5002 (PMI Nutrition International, Inc., St. Louis, MO), ad libitum
- Water: automatic watering system, Municipal water supply (Elizabethtown Water Company), ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 – 26
- Humidity (%): 30 -70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED: 2 mL/kg

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

other: not required

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: animals were checked for viability daily. Each animal was examined (general condition, skin and fur, eyes nose, oral cavity, abdomen and external genitalia as well as evaluations of respiration and palpation for tissue masses) approximately 1, 2, and 4 hours after dosing and daily thereafter for 14 days. Individual body weights were determined on day 0 (at the time of fasting), day 1 (just prior to dosing; weights were used for calculation of doses), day 8 and 15
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, food consumption, macroscopic post-mortem examination.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occurred during the study period.

Clinical signs:

other: No clinical signs of toxicity related to the administration of the test substance were observed up to the end of the 14-day observation period. However alopecia extremities on snout was seen in a single animal on day 9 through day 15 after the dose admini

Gross pathology:

Necropsy revealed no substance-related findings.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Reference 4

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

yes

Remarks:

: Only two male and two female animals used

GLP compliance:

not specified

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: Alderley Park SPF albino

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: males: 215 g - 237 g; females: 145 g - 163 g
- Fasting period before study: 24 h

Route of administration:

oral: unspecified

Vehicle:

corn oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 200 mg/mL

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg

Doses:

2000 mg/kg

No. of animals per sex per dose:

2

Control animals:

no

Details on study design:

- Frequency of observations and weighing: daily
- Necropsy of survivors performed: yes
- Duration of observation period following administration: not stated
- Other examinations performed: body weight, macroscopic abnormalities (post mortem)

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occurred during the study period.

Clinical signs:

other: No significant signs of toxicity in females but males showed slight toxicity following a 2000 mg/kg bw dose.

Gross pathology:

Necropsy and histopathological examination revealed no substance-related findings.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Reference 5

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Japan
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: mean 201 g
- Fasting period before study: 16 h priot to dosing
- Housing: 1 - 3 animals per cage
- Diet: MF solid diet (Oriental Yeast Production (Lot No.040705, 041102), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 9-16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.5 - 24.2
- Humidity (%): 48 - 81
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Doses:

300 and 2000 mg/kg bw

No. of animals per sex per dose:

6

Control animals:

other: not required

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: observations every six hours on administration day starting one hour after administration, then daily for 14 days; weighing prior to dosing and on days 7 and 154
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No deaths were observed with either 300 and 2000 mg/kg bw during the observation period.

Clinical signs:

other: Diarrhea was observed in one animal receiving 300 mg/kg bw on the administration day.

Gross pathology:

No abnormalities were noted in any animals.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Reference 6

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 Mar - 4 Nov 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

yes

Remarks:

no analytical purity; method shortly described

GLP compliance:

no

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms
- Fasting period before study: overnight
- Housing: individually in single wire bottom stainless steel suspended cages
- Diet: Purina Dawley Chow # 5002
- Water: Automatic watering; ad libitum

Route of administration:

oral: gavage

Doses:

15000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

other: not required

Details on study design:

- Duration of observation period following administration: 14 days
- Necropsy of survivors performed: no
- Other examinations performed: clinical signs

Sex:

male/female

Dose descriptor:

LD50

Effect level:

>= 15 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occurred during the study period.

Clinical signs:

other: No clinical signs of toxicity related to the administration of the test substance were observed up to the end of the 14-day observation period. However, all animals exhibited clear, oily perineal staining on the 1st, 2nd, and 3rd day following treatment;

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises adequate and reliable (Klimisch score 1 and 2) studies from various source substances with similar structures and intrinsic properties. Read-across is justified based on common precursors and hydrolysis products and consistent trends in environmental fate, ecotoxicological and toxicological profile. The selected studies are thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.5, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24 Jul - 8 Dec 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

yes

Remarks:

no analytical purity reported

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Kingston, NY, USA
- Age at study initiation: 8 weeks
- Housing: individually in stainless wire mesh cages.
- Diet: certified Purina rodent chow #5002, ad libitum (except during exposures)
- Water: tap water (delivered via automatic system except during exposures)
- Acclimation period: minimum of 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 40-60
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel exposure chamber
- Exposure chamber volume: 400 L
- Method of holding animals in test chamber: Animals were housed in a nearby room and loaded into cages designed for the inhalation chamber for exposure
- Source and rate of air: Pressurized air passed through the hollow stream of nebulizer and exited at high velocity through holes in its side. This high velocity air stream passed over the top of the hollow feed barrels and caused the aspiration of the liquid test material up into the feed barrel. The liquid was aerosolized as it was drawn into the airstream by the relative negative pressure there.
- System of generating particulates/aerosols: Laskin nebulizers
- Method of particle size determination: The mass median aerodynamic diameter of the aerosol was determined once during exposure by use of a cascade impactor
- Temperature, humidity, pressure in air chamber: 24.4 °C (control group) and 23.8 °C (0.48 and 4.06 mg/L groups); 60% (control and 4.06 mg/L group), 64% (0.48 mg/L)

TEST ATMOSPHERE
- Brief description of analytical method used: The concentration of aerosol in the exposure chamber was determined gravimetrically by drawing known volumes of air from the chamber through glass fibre filters and measuring the increase in weight of the filters. The weight increase was divided by volume of air sampled to give aerosol concentration.
- Samples taken from breathing zone: no

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution:
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 0.9/1.9 (0.48 mg/L) and 1.1/1.7 (4.06 mg/L)

Analytical verification of test atmosphere concentrations:

yes

Remarks:

gravimetric

Duration of exposure:

ca. 4 h

Concentrations:

0.48 or 4.06 mg/L (analytical concentration)
0.60 or 4.07 mg/L (nominal concentration)

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: individual body weight were recorded on days 1 (day of first exposure), 2, 9, and 17. Clinical signs were recorded daily except of weekends.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, organ weights, histopathology

Statistics:

Data on body weights were analyzed by ANOVA and Tukey´s multiple range test for comparison of control and exposed groups.

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 4.06 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No mortality occurred during the study period.

Clinical signs:

other: No clinical signs of toxicity were observed up to the end of the 14-day observation period.

Body weight:

No effect on body weight was noted.

Gross pathology:

Necropsy examination revealed no substance-related findings.

Other findings:

- Organ weights: No treatment-related changes were observed in the weights of the liver or kidneys. Both wet and dry weight of the right middle lung lobe tended to be higher in the exposed males at 2 weeks post-exposure. Dry weight was significantly greater in group 3 relative to group 1, but only in males at 2 weeks.
- Histopathology: Histopathological examination revealed no substance-related findings. Since no treatment-related morphologic changes were found at macroscopic post mortem examination of the animals, without this morphologic confirmation, the small changes in lung weight were considered not to be biologically relevant.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Reference 2

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

adopted in 1996

Deviations:

yes

Remarks:

analytical purity not reported

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: Sprague-Dawley CD®

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Klingston, NY, USA
- Age at study initiation: approximately 9 weeks (control group); approximately 10 weeks (test group)
- Weight at study initiation: 332 g (control group, males only); 358 g (test group, male) and 247 (test group, female)
- Fasting: feed was not provided during the exposure
- Housing: animals were group-housed in suspended, stainless steel, wire mesh cages during the acclimation period and individually-housed during all other non-exposure period.
- Diet: certified Rodent Diet, # 5002 meal, ad libitum
- Water: ad libitum
- Acclimation period: The control group animals were acclimated for 12 days. The test group animals were acclimated for 19 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 18-30
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: cast aluminium and alloy exposure chamber with polycarbonate nose-only tubes housed within 10 m³ Harford glass and stainless steel exposure chamber.
- Exposure chamber volume: 40 L
- Method of holding animals in test chamber: animals were placed in polycarbonate tubes attached to the chambers.
- Source and rate of air: 20 L/min
- System of generating particulates/aerosols: Approximately 110 mL of test material were placed into a nebulizer. House-supply air was delivered from a regulator and backpressure gauge, via ¼” tubing, to a plastic Y tube which split the airflow into the generation and dilution systems. The generation air (7.0 Lpm) was directed, via ¼” tubing, through a flowmeter regulated by a metering valve, and a backpressure gauge, to the nebulizer. The test material-laden airstream was directed through a flowmeter regulated by a metering valve, into the dilution port at the top of the nose-only exposure chamber.
- Method of particle size determination: In the control group the particle size distribution measurements were performed each hour of exposure for the chamber and room air using a TSI Aerodynamic Particle Sizer. The samples were drawn for 20 seconds at a rate of 5.0 Lpm. The mass median aerodynamic diameter, geometric standard deviation and in the test group samples for particle size distribution assessment were drawn each hour of exposure using a cascade impactor. The samples were drawn for one minute at a flowrate of 1.00 Lpm. The mass median aerodynamic diameter, geometric standard deviation and percent of particles ≤ 1.0, ≤ 4.0 and ≤ 10 microns were calculated based on the amount of material collected on the seven impactor stages and a final filter stage using a graphical analysis of an assumed lognormal distribution.
- Temperature, humidity, pressure in air chamber: 21°C (average), 23% (average)

TEST ATMOSPHERE
- Brief description of analytical method used: Sample concentration (mg/L) was determined gravimetrically on a total formulation basis. A drop of the test material was placed on a weighed filter, weighed immediately and again after drying overnight. This was done in triplicate. The resultant data were used to determine the fraction of solids in the test material. During the exposure, the filter was weighed before and immediately after sampling (wet weight). The exposure concentration (mg/L) was calculated on both a “wet” and “dry” basis by dividing the appropriate net weight (mg) by the volume of air sampled (liters). For the test group, only exposure levels were calculated on a formulation by dividing the exposure concentration on a “dry” basis (mg/L) by the fraction of solids.
- Samples taken from breathing zone: yes

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution:
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 2.18/2.03

Analytical verification of test atmosphere concentrations:

yes

Remarks:

gravimetric

Duration of exposure:

4 h

Concentrations:

5.50 mg/L (analytical concentration)
6.6 mg/L (nominal concentration)

No. of animals per sex per dose:

10 (air control)
10 (males, test group)
5 (females, test group)

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days (5 males were sacrificed on Day 3)
- Frequency of observations and weighing: on day 1 all animals were observed individually immediately prior to the exposures, as a group at approximately 15 minute intervals during the first hour of each exposure, and hourly for the reminder exposure periods. All surviving animals were observed individually upon removal from the chambers (30 min after each exposure termination) and every hour for two hours post-exposure. Detailed physical observations (general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia as well as evaluations of respiration and palpation for tissue masses) were recorded at each interval. From day 2 to 15 detailed observations were recorded once daily. Individual body weights were recorded on day 1 (just prior to exposure), on Day 3 (just before sacrifice). Animal sacrificed on Day 15 were weighed on day 8 and 15 (just prior sacrifice)
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, histopathology, gross pathology.

Sex:

male/female

Dose descriptor:

LC50

Effect level:

5.5 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No mortality occurred during the study period.

Clinical signs:

other: Test group: nasal discharge was noted as only sign of toxicity. Wet fur was noted as well but was considered an artefact of the nose-only exposure regimen. A few incidences of chromodacryorrhea were also noted to a comparable degree to the Group II animal

Body weight:

The test group females lost weight during the first week after the test material exposure, but gained weight during the second week after exposure. In all other animals no effect on body weight was noted.

Gross pathology:

Necropsy examination revealed no substance-related findings.

Other findings:

- Histopathology: There were no test material related microscopic findings. At the end of each of the post-exposure observation periods, incidental findings occurred with comparable incidence and severity in rats from the control and test groups or they occurred sporadically. The have been seen of this strain and age used in similar studies conducted in the study facility.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Reference 3

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

22 Mar - 05 Apr 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

yes

Remarks:

(No details on test material and limited data on animal husbandry)

GLP compliance:

not specified

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: Alpk:APfSD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Alderly Park, Cheshire, UK
- Age at study initiation: approx. 7 weeks

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Source and rate of air: dried and filtered air
- System of generating aerosols: glass concentric jet atomiser
- Method of particle size determination: Marple Cascade Impactor
- Temperature, humidity, pressure in air chamber: 20 L/min

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetrically
- Samples taken from breathing zone: yes

TEST ATMOSPHERE (if not tabulated)
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 1.51 µm/2.51

Analytical verification of test atmosphere concentrations:

yes

Remarks:

particulate concentrations of the test atmospheres close to the animals breathing zone were measured gravimetrically

Duration of exposure:

4 h

Concentrations:

5.0 mg/L (nominal)
5.10 mg/L (analytical)

No. of animals per sex per dose:

5

Control animals:

not specified

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: animals were observed for gross clinical abnormalities during exposure and were checked daily thereafter. A detailed examination was conducted after exposure on day 1 and on consecutive days, up to and including day 15. Individual body weights were recorded on Day 1 and Days 2, 3, 8 and day 15.
- Necropsy of survivors performed: yes

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.1 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No mortality occurred during the study period.

Clinical signs:

other: Some clinical signs were noticed, which consisted of hunched position, chromodacryorrhea, piloerection, staining around nose and wet fur. These signs however occurred during or just after exposure and were clearly consistent with the use of restraint for

Body weight:

No effect on body weight was noted.

Gross pathology:

Necropsy and histopathological examination revealed no substance-related findings.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Reference 4

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

22 Mar - 05 Apr 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

yes

Remarks:

Limited data on study substance

GLP compliance:

not specified

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: Alpk:APfSD

Sex:

male/female

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

not specified

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Not stated
- Source and rate of air: 20 L/min using a peristaltic pump
- Method of conditioning air: Clean dry air (dried and filtered, no further details)
- System of generating particulates/aerosols: Glass concentric jet atomiser
- Method of particle size determination: Marple Cascade Impactor

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetrical measurement
- Samples taken from breathing zone: yes

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: No Data
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 1.76 µm / 2.35 µm

Analytical verification of test atmosphere concentrations:

yes

Remarks:

Gravimetrically measurement of particulate concentrations

Duration of exposure:

4 h

Concentrations:

5 mg/L

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations: daily
- Frequency of weighing: on Days 1, 2, 3, 8 and 15
- Necropsy of survivors performed: yes

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No mortality occurred during the study period.

Clinical signs:

other: Clinical signs seen during and/or immediately after exposure were hunched posture, chromodacryorrhea, piloerection, stains around the nose and wet fur. In general, animals showed a rapid recovery from these effects by Day 2, although, hunched posture and

Body weight:

No effect on body weight was noted.

Gross pathology:

Necropsy and histopathological examination revealed no substance-related findings.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises adequate and reliable (Klimisch score 2) studies from various source substances with similar structures and intrinsic properties. Read-across is justified based on common precursors and hydrolysis products and consistent trends in environmental fate, ecotoxicological and toxicological profile. The selected studies are thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.5, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

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:

05 Jan - 19 Jan 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, UK

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: Sprague-Dawley CD (Crl : CD® BR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Ltd, Margate, Kent, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 211 – 225 g (males) and 208 – 232 g (females)
- Housing: animals were housed individually during the 24-h exposure and in groups of five of the same sex, for the remainder of the study, in polypropylene cages furnished with woodflakes.
- Diet: Rat and Mouse SQC Expanded Diet No. 1 (Special Diets Services Limited, Witham, Essex, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Example: 18-21
- Humidity (%): 47-67
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

OTHER
- On one occasion the temperature was below the limit specified in the protocol (19 °C). This deviation was considered not to affect the purpose or integrity of the study.

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: back and flanks were clipped
- % coverage: 10%
- Type of wrap if used: The test substance was held in contact with the skin with surgical gauze and semi-occluded with a piece of self-adhesive bandage.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Residual test material was removed with cotton wool moistened with distilled water.
- Time after start of exposure: 24 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):2.06 mL/kg bw
- Concentration (if solution): 100%

Duration of exposure:

24 h

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

not required

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: animals were observed 30 min, 1, 2 and 4 h after dosing and subsequently daily for 14 days and individual body weights were determined prior to dosing and 7 and 14 days after treatment.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occurred during the study period.

Clinical signs:

other: No clinical signs of toxicity were observed up to the end of the 14-day observation period.

Gross pathology:

Necropsy revealed no substance-related findings.

Other findings:

Dermal reactions:
No signs of skin irritation were noted during the study period in any animal.

Table 1. Individual body weights and weekly body weight changes.

Dose level mg/kg bw	Animal number and sex	Bodyweight (g) at Day			Body weight gain (g) during week
		0	7	14	1	2
2000	1-0 Male	225	267	319	42	52
	1-1 Male	215	259	307	44	48
	1-2 Male	217	255	291	38	36
	1-3 Male	211	253	300	42	47
	1-4 Male	221	248	286	27	38
	1-0 Female	208	219	226	11	7
	1-1 Female	210	210	231	0	21
	1-2 Female	232	246	257	14	11
	1-3 Female	208	210	217	2	7
	1-4 Female	213	216	220	3	4

 

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises an adequate and reliable (Klimisch score 1) study from a source substance with similar structures and intrinsic properties. Read-across is justified based on common precursors and hydrolysis products and consistent trends in environmental fate, ecotoxicological and toxicological profile. The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.5, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

Additional information

Justification for grouping of substances and read-across

The read-across from analogue source substances approach comprises aliphatic esters of poly-functional alcohols containing two to six reactive hydroxyl groups and one to six fatty acid chains. The analogue approach contains mono constituent, multi-constituent and UVCB substances with fatty acid carbon chain lengths ranging from C5 - C28, which are mainly saturated but also mono unsaturated C16 and C18, polyunsaturated C18, branched C5 and C9, branched C14 - C22 building mono-, di-, tri-, and tetra esters with an alcohol (i.e. the polyol).

The available data allows for an accurate hazard and risk assessment of the target substance and the read-across concept is applied for the assessment of environmental fate and environmental and human health hazards. Thus, where applicable, environmental and human health effects are predicted from adequate and reliable data for source substances within the group by interpolation to the target substance applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No. 1907/2006. In particular, for each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across.

A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Section 7.1 and 13).

Acute oral toxicity

There are several reliable acute oral toxicity studies available for structural analogue source substances, all of which are taken into account by means of a Weight-of-Evidence approach.

The acute oral toxicity of Dipentaerythritol hexaesters with fatty acids, C5 and C9iso (CAS No. 647028-25-9) was investigated in a study according to OECD Guideline 423 performed under GLP conditions (WoE, RA-A, 647028-25-9, 1999a). Groups of 3 female and male Crl:CD (SD) rats were dosed at 2000 mg/kg bw and observed for 14 days after treatment. No mortality occurred during the study period. No clinical signs of toxicity or effects on body weights were observed until the end of the observation period. Necropsy revealed no substance-related findings. The oral LD50 value for male and female rats was therefore determined to be > 2000 mg/kg bw.

The surrogate substance Hexanoic acid, 2-ethyl-, 2,2-bis [ [(2-ethyl-1-oxohexyl)oxy] methyl] -1,3-propanediyl ester (CAS No. 7299-99-2) was tested in an OECD 423 study in compliance with GLP (MHLW, 2005a). A dosage of 300 and 2000 mg/kg bw was administered by gavage to groups of six female Crj: CD(SD) rats, respectively. Diarrhoea was observed in one animal receiving 300 mg/kg bw on the administration day. However, no other clinical signs, no effects on body weight gain, and no mortality were observed during the 14-day observation period. Therefore, a LD50 of > 2000 mg/kg bw was determined.

A study performed according to OECD Guideline 401 investigated the acute oral toxicity of Pentaerythritol ester of pentanoic acids, mixed esters with pentaerythritol, isopentanoic and isononanoic acid (CAS No. 146289-36-3) (Emery, 1991). 5 male and 5 female Wistar rats were administered a single dose of 2000 mg/kg bw of the test substance by gavage. No mortality occurred during the study period. No clinical signs of toxicity were observed up to the end of the 14-day observation period and no effect on body weights was noted. Necropsy and histopathological examination revealed no substance-related findings. Therefore, the oral LD50 value in male and female rats was found to exceed 2000 mg/kg bw.

The study conducted with Fatty acids, C5-10, esters with pentraerythritol (CAS No. 68424-31-7) (WoE, RA-A, 68424-31-7, 1991a) with limitations (reduced animal number), revealed no mortality and no other toxic effects, but an initial weight loss after treatment of rats with 2000 mg/kg bw. However this effect was completely reversible and the animals showed subsequently normal body weight gain. The oral LD50 value was therefore determined to exceed 2000 mg/kg bw.

The acute oral toxicity of Fatty acids, C5-9 tetraesters with pentaerythritol (CAS No. 67762-53-2) has been investigated in two studies with rats. In the first study (Exxon, 1999a), groups of 5 male and female fasted CD Sprague-Dawley rats received single oral gavage doses of 2000 mg/kg bw. The animals were observed for 14 days following administration. No mortalities occurred. One animal showed alopecia extremities on snout which was not considered treatment related. No further clinical signs of toxicity were reported. No effect on body weight was noted. Terminal necropsy revealed no substance-related findings. Thus, the acute oral LD50 was found to be greater than 2000 mg/kg bw.

Another acute oral toxicity study (limit test) was performed comparable to OECD Guideline 401 (Exxon, 1984). The test substance was administered by gavage at a concentration of 15000 mg/kg bw to groups of 5 male and female Sprague-Dawley rats. The animals were observed for 14 days following administration. No mortalities occurred. All animals exhibited clear, oily perineal staining on the 1st, 2nd, and 3rd day following treatment. This condition normally regarded as an adverse effect, was considered to be a consequence of the high dose level administered and not considered a sign for toxicity. No further clinical signs of toxicity were reported. The acute oral LD50 was found to be greater than 15000 mg/kg bw.

The source substance Pentaerythritol ester of pentanoic acids, mixed esters with pentaerythritol, isopentanoic and isononanoic acid (CAS No. 146289-36-3) was investigated regarding its acute oral toxicity. A limit test according to OECD Guideline 401 and compliant to GLP provisions was performed (Emery, 1991). 5 male and 5 female Wistar rats were administered 2000 mg/kg bw of the test substance by gavage and observed for a period of 14 days after treatment. No mortality occurred during the study period. No clinical signs of toxicity and no effects on body weights were observed until the 14-day observation period. Moreover, histopathological examination during terminal necropsy revealed no substance-related findings. An LD50 value of > 2000 mg/kg bw was therefore determined in this study.

Acute inhalation toxicity

Several reliable and adequate studies are available regarding the acute inhalation toxicity of structural analogue source substances. The studies are accounted for in a Weight-of-Evidence approach.

An acute inhalation toxicity study was performed with Fatty acids, C5-10, esters with pentaerythritol (CAS No. 68424-31-7) comparable to OECD Guideline 403. 5 male and female Alpk:APfSD rats were exposed for 4 hours to 5.0 mg/L air test substance aerosol by nose only inhalation (WoE, RA-A, 68424-31-7, 1994a). The test substance caused no mortality, body weight changes or abnormalities in necropsy during the 15 day study period. Some clinical signs were noticed, which consisted of hunched position, chromodacryorrhea, piloerection, staining around nose and wet fur. These signs however occurred during or just after exposure and were clearly consistent with the use of restraint for exposure. The LC50 was therefore found to be greater than 5.1 mg/L.

Two studies investigating the acute toxicity via the inhalation route of exposure are available for the source substance Fatty acids, C5-9 tetraesters with pentaerythritol (CAS No. 67762-53-2). The first study was conducted comparable to OECD Guideline 403 and according to GLP. 10 male and female Sprague-Dawley rats were exposed for 4 hours to 0.48 or 4.06 mg/L test substance aerosol by whole body inhalation exposure (Exxon, 1990). No mortality, clinical signs, body weight changes or abnormalities in necropsy were observed during the 15 day study period in any group. The LC50 was therefore found to be greater than 4.06 mg/L.

In the second study 10 male and 5 female CD Sprague-Dawley rats were exposed for 4 hours to 5.5 mg/L test substance aerosol by nose/head only inhalation (Exxon, 1999b). No mortalities occurred during the study period. Nasal discharge was noted as the only sign of clinical toxicity. The test group females lost weight during the first week after the test material exposure, but gained weight during the second week after exposure. In all other animals no effect on body weight was noted. Necropsy examination revealed no substance-related findings. The LC50 was therefore found to be greater than 5.50 mg/L.

An acute inhalation toxicity study was performed with Fatty acids, C5-9, mixed esters with dipentaerythritol and pentaerythritol (CAS No. 85536-35-2) comparable to OECD Guideline 403. 5 male and female Alpk:APfSD rats were exposed for 4 hours to 5.0 mg/L air test substance aerosol by nose only inhalation (WoE, RA-A, 85536-35-2, 1994b). The test substance caused no mortality, body weight changes or abnormalities in necropsy during the 15 day study period. Clinical signs during and immediately after exposure included hunched posture, chromodacryorrhea, piloerection, stains around the nose and wet fur. In general, animals showed a rapid recovery from these effects by Day 2, although, hunched posture and piloerection persisted in few animals to Days 4 and 8, respectively. No effect on body weight was noted. Necropsy and histopathological examination revealed non substance-findings. The LC50 was therefore found to be greater than 5.0 mg/L air.

Acute dermal toxicity

Acute dermal toxicity has been studied using a structural analogue source substance.

Dipentaerythritol hexaesters with fatty acids, C5 and C9iso (CAS No. 647028-25-9) has been investigated for its acute dermal toxicity in a study according to OECD Guideline 402 and observing GLP provisions (Key, RA-A, 647028-25-9, 1999b). The unchanged test substance was applied to the clipped backs and flanks of 5 male and 5 female Crl:CD (SD) rats under semiocclulsive conditions. The exposure period was 24 h before residual test material was removed with cotton wool moistened with distilled water. After removal of the dressings and subsequently once daily during the 14-day observation period, the test sites were examined for evidence of primary irritation and scored according to the Draize scoring system. No mortality occurred during the study period. No clinical signs of toxicity and no effects on body weights were observed until the end of the observation period. Terminal necropsy revealed no substance-related findings. Moreover, no signs of skin irritation were noted during the study period in any animal. The acute dermal LD50 value was therefore determined to be > 2000 mg/kg bw.

Conclusion on acute toxicity

Several reliable and adequate studies performed with analogue source substances are available investigating the acute oral, inhalation and dermal toxicity of different structural analogue polyol esters resulting in oral LD50 values of > 2000 mg/kg bw, inhalation LC50 values of > 5.0 mg/L air and a dermal LD50 value of > 2000 mg/kg bw. The available data indicate a very low level of acute toxicity for the analogue source substances and thus, no hazard for acute oral, inhalation and dermal toxicity is identified for the target substance Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids as well.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 information on intrinsic properties of substances may be generated by means other than tests, e.g. using information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the read-across concept is applied to the target substance Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids, data gaps can be filled by interpolation from representative structural analogue source substances to avoid unnecessary animal testing.

The read-across concept is also used to derive the classification of the target substance taking the properties of the source substances into account. Based on the read-across concept, all available data on acute toxicity do not meet the classification criteria according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.