Castor oil, ester with glycerol

Endpoint summary

Description of key information

In the absence of skin sensitisation data on target substance Castor oil, ester with glycerol an analogue read-across approach was conducted on suitable source substances.

Skin sensitisation (QSAR target substance; GPMT, LLNA source substances): not sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18 Apr - 24 Apr 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, ländlichen Raum und Verbraucherschutz, Wiesbaden, Germany

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA7CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, Horst, The Netherlands
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 19.4 ± 0.9 g
- Housing: animals were housed individually in Makrolon Type I cages with wire mesh top (EHRET GmbH, Emmedingen, Germany) and granulated soft wood bedding (Harlan Winkelmann GmbH, Borchen, Germany)
- Diet: pelleted standard diet (Harlan Winkelmann GmbH, Borchen, Germany), ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

dimethyl sulphoxide

Concentration:

10, 20 and 50% (w/v)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Study design: 50% (Batch D) was tested in a non-GLP pre-experiment
- Compound solubility: insoluble in water, soluble in DMSO
- Lymph node proliferation response: DPM-Background (1 mL 5% trichloroacetic acid) per animal (2 lymph nodes) controls: 2707.1 (animal 1), 2357.9 (animal 2), 2199.6 (animal 3), 2336.3 (animal 4) and 3184.5 (animal 5); DPM-Background (1 mL 5% trichloroacetic acid) per animal (2 lymph nodes) test group: 6794.2 (animal 1), 5977.4 (animal 2), 6981.8 (animal 3), 7739.5 (animal 4), 6305.7 (animal 5)

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³H-methyl thymidine incorporation determined by β-scintillation and γ-counting, respectively
- Criteria used to consider a positive response: A test item was regarded as a sensitiser in the LLNA if the following criteria were fulfilled: The exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index. The data were compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression. The decision to select a stimulation index (SI) of 3 as an arbitrary indication of sensitising activity was made on the basis of investigations performed with a wide range of chemicals.

TREATMENT PREPARATION AND ADMINISTRATION: Each test group was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations. The application volume, 25 µL, was spread over the entire dorsal surface of each ear lobe once daily for 3 consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). Five days after the first topical application, all mice were administered with 250 µL of 80.6 µCi/mL ³HTdR (corresponds to 20.2 µCi ³HTdR per mouse) by intravenous injection via a tail vein. Five hours later, all mice were euthanized by intraperitoneal injection of Na-thiopental. The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions in PBS of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with PBS the lymph node cells were resuspended in 5% trichloroacetic acid and incubated at approximately 4 °C for at least 18 h for precipitation of macromolecules. The precipitates were resuspended in 5% trichloroacetic acid and transferred to plastic scintillation vials.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean values and standard deviations were calculated in the body weight tables. An ANOVA (Kruskal Wallis) followed by a Dunnett-test or t-test (Mann-Whitney), when applicable, were conducted to assess whether the difference of DPM per animal was statistically significant between test item groups or the positive control (RCA) group and the negative control (vehicle) group. However, both biological and statistical significance were considered together.

Positive control results:

The positve control substance (25% hexyl cinnamic aldehyde in acetone:olive oil (4+1)) induced positive reactions in 2/5 animals (40%).The SI-values of the positive control were 2.3, 1.6, 2.6, 4.2 and 4.8, respectively.

Key result

Parameter:

SI

Value:

0.97

Test group / Remarks:

10%

Remarks on result:

other: maximum SI of all batches

Key result

Parameter:

SI

Value:

1.53

Test group / Remarks:

25 %

Remarks on result:

other: maximum SI of all batches

Key result

Parameter:

SI

Value:

1.86

Test group / Remarks:

50%

Remarks on result:

other: maximum SI of all batches

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
At a concentration of 50% of Batch A a statistically significant difference of DPM per animal was observed (p ≤ 0.05), however, the stimulation index (SI) was 1.71. All other test groups did not show a statistically significant different response from the vehicle control. The effects observed for Batch A seemed to be dose-dependent. The effects observed for Batch B and D seemed to be dose-dependent but this was not confirmed by statistical analysis. The effects seen for Batch C were not dose-dependent.

EC3 CALCULATION
EC3 values of the test groups could not be calculated, since none of the tested concentrations induced a SI greater than 3.

CLINICAL OBSERVATIONS:
The animals treated with the mid- and the highest-dose (25% and 50%) of Batch A showed reddening of the ear skin from the second day of treatment. The animals treated with Batch B, C and D did not show any clinical signs during the course of the study and no cases of mortality were observed.

VEHICLE CONTROL DATA:

DPM values per lymph node obtained for the vehicle control were relatively high when compared to the historical control data. The results for the individual animals showed low variability and the results are nonetheless considered as reliable. Differences in vehicle control data when compared to other studies are related to the use of different batches of 3H-Thymidine. Experiments performed with the same batch of 3H-Thymidine as used in the present study have shown DPM values per lymph node, which were considerably higher than the results obtained with earlier and later batches. It is assumed that incorporation of 3H-Thymidine is more efficient with the present batch even though evidence for this hypothesis cannot be provided. Since all animals used for this study were labeled with the same 3H-Thymidine-solution this increase is supposed to be higher for all test groups and does neither affect the sensitivity of the assay nor the calculation of S.l. values.

Table 1. Results of the LLNA.

Test item concentration % (w/v)	Group	Calculation	DPM per lymph node	Result
		number of lymph nodes		SI
-	BG_I		37.1*
-	BG_II		17.89*
-	Control group	10	2027.9	1.0
25	Positive control Group	10	6312.4**	3.11
10	Test group Batch A	10	1528.3	0.75
25	Test group Batch A	10	2088.3	1.03
50	Test group Batch A	10	3468.7**	1.71
10	Test group Batch B	10	1964.4	0.97
25	Test group Batch B	10	3107.2	1.53
50	Test group Batch B	10	3768.4	1.86
10	Test group Batch C	10	1211.2	0.60
25	Test group Batch C	10	2295.3	1.13
50	Test group Batch C	10	2213.7	1.09
10	Test group Batch D	10	1225.7	0.60
25	Test group Batch D	10	1688.8	0.83
50	Test group Batch D	10	2868.6	1.41

* Measurement of DPM only.

** Mean DPM value for the group was according to the Dunnett test significanlty higher than the corresponding control value. The p value for the analysis was <0.05

BG = Background (1 mL 5% trichloroacetic acid) in duplicate

SI = Stimulation Index

Interpretation of results:

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

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11 May - 10 Sep 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted in 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

adopted in 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Type of study:

Buehler test

Justification for non-LLNA method:

The Buehler Test was performed prior to the amendment of Regulation (EC) No 1907/2006 in which the Local Lymph Node Assay is given as the first-choice in vivo study.

Species:

guinea pig

Strain:

other: Dunkin-Hartley, Pirbright White Hsd/Poc:DH

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany.
- Age at study initiation: young adults
- Weight at study initiation: < 500 g
- Housing: maximum 5 animals per cage in Type IV Makrolon cages.
- Diet: Ssniff G 4 diet in pellet form (laboratory standard guinea pig dietdiet, Ssniff Spezialfutter GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70 (temporary deviations were caused by cleaning the animal room)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

50%

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

50%

No. of animals per dose:

10 (controls), 20 (in test groups)

Details on study design:

RANGE FINDING TESTS:
In a preliminary miscibility test, a test substance concentration of 50% (w/w) was determined as the highest concentration which was easily miscible and well applicable.
In a preliminary skin irritation test with 3 female animals, test substance formulations of 10, 20, 30 and 50% were topically applied to the flank under occlusive conditions for 6 h. The maximum non-irritant concentration of 50% was used for challenge.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 h
- Test groups: test substance in vehicle
- Control group: vehicle
- Site: left flank
- Frequency of applications: every 7 days
- Duration: Days 0-14
- Concentrations: 50% test substance in vaseline

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 28
- Exposure period: 6 h
- Test groups: test substance in vehicle and vehicle only
- Control group: test substance in vehicle and vehicle only
- Site: posterior right flank (test substance) and anterior right flank (vehicle)
- Concentrations: 50% test substance in vaseline
- Evaluation (hr after challenge): 24 and 48 h after test substance removal with corn oil.

Challenge controls:

In the 4th week of the test, 3 additional animals (accompanying group), kept under the same conditions, but without treatment, were used to re-determine the maximum non-irritant concentration for the challenge treatment.
This additional determination of the challenge concentration was carried out because it was suspected that the sensitivity of the skin changed as the weight of the animals increased. This ensured that the challenge concentration was determined on animals which had approximately the same weight as the 30 animals in the challenge phase. In this test, the concentrations administered and the experimental conditions corresponded to those of the preliminary test.
The determination of the challenge concentration in the 4th week of the test on 3 untreated animals of the same age led to no signs of dermal irritation in any animal 24 and 48 h post-application of formulations containing 10, 20, 30 and 50% test substance in vehicle.
On the basis of these results and the results of the induction period, the 50% test substance in vehicle was administered for the challenge treatment in the main test.
Since this test was conducted with untreated animals approximately at the same time as the challenge application, it can be considered as a challenge control test.

Positive control substance(s):

yes

Remarks:

α-hexylcinnamaldehyde

Positive control results:

The positive control substance α-hexylcinnamaldehyde (100% induction, 50% challenge concentration) induced positive reactions in 10/20 and 9/20 animals at 24 and 48 h after challenge, respectively. Thus, the reliability criteria for the Buehler test (≥ 15% positive response) were met.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

20

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

100% induction, 50% challenge

No. with + reactions:

10

Total no. in group:

20

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

20

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

100% induction, 50% challenge

No. with + reactions:

9

Total no. in group:

20

Interpretation of results:

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

Reference 3

Endpoint:

skin sensitisation, other

Remarks:

QSAR

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
The OECD QSAR Toolbox v4.0 is a Quantitative Structure-Activity Relationship model that was developed by the Laboratory of Mathematical Chemistry (http://toolbox.oasis-lmc.org).

2. MODEL (incl. version number)
OECD QSAR Toolbox, version 3.3

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
SMILES codes for representative constituents of the test substance were used (see 'Test material information for details').

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: The model was used to predict the skin sensitisation potential of the main constituents of the test substance in the database 'Protein binding alerts for skin sensitisation by OASIS v1.4'. The profiler has been developed by industry consortia with the Laboratory of Mathematical Chemistry and the partnership of Dr D.Roberts, as a part of the TIMES model for predicting skin sensitization. The profiler is intended to be used for the assessment of protein binding interaction of chemicals and especially interaction with skin proteins. The profiler has been developed based on mechanistic knowledge for skin sensitisation of dataset of 875 chemicals tested by Local Lymph Node Assay (LLNA), Guinea Pig Maximization Test (GPMT) and chemicals from the BfR list. A list of 100 structural alerts has been derived, based on the mechanistic knowledge of training set chemicals. The scope of the profiler is to investigate the presence of alerts within the target molecules responsible for interaction with proteins and especially with skin proteins.
- Defined domain of applicability: This profiler accounts for incapability of some chemicals having an alert to interact with skin due to electronic and steric factors. This is explicitly defined by inhibition masks associated with some alerts. The list of 100 structural alerts has been separated into 11 mechanistic domains. Each of the mechanistic domains has been separated into more than 2 mechanistic alerts. The profiling result outcome assigns a target to the corresponding structural alert, mechanistic alerts and domain.

5. APPLICABILITY DOMAIN
- Descriptor domain: The profiler is applicable to those organic chemicals that have presence of at least one of the 100 protein binding alerts specified within the profiler. The presence of protein binding alerts is not bounded with parametric ranges; it is based on structural boundaries only. The absence of a structural alert should not be taken as an absence of toxicity. The test substance did not show any alerts.
- Similarity with analogues in the training set: The profiler has been developed based on mechanistic knowledge for skin sensitisation of a dataset of 875 chemicals. Therefore the relevance of the training set is that similarity may indicate a skin sensitising potential and lack of similarity (alerts), as for the test substance indicates a lack of skin sensitising potential.

6. ADEQUACY OF THE RESULT
The results are considered in a weight-of-evidence approach together with other information to reach a conclusion regarding the skin sensitising potential of the test substance.

6. ADEQUACY OF THE RESULT
The results may be used in a weight-of-evidence approach together with other information to reach a conclusion regarding the skin sensitising potential of the test substance.

Principles of method if other than guideline:

- Principle of test: The OECD QSAR Toolbox v4.0 is a Quantitative Structure-Activity Relationship model that was developed by the Laboratory of Mathematical Chemistry (http://toolbox.oasis-lmc.org). It contains several different databases with data on chemicals. The model was used to predict the skin sensitisation potential of the main constituents of the test substance in the database 'Protein binding alerts for skin sensitisation by OASIS v1.4'.
- Short description of test conditions: The SMILES code of the main constituents of the test substance is compared with funtional groups known to be related to potential skin sensitising properties.
- Parameters analysed / observed: A QSAR prediction of the skin sensitisation potential of the main constituents of the test substance was performed. The presence of protein binding alerts that may indicate a skin sensitising potential is assessed.

GLP compliance:

no

Key result

Run / experiment:

other: Skin sensitisation prediction as structural alerts

Parameter:

other: QSAR prediction

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: Negative for skin sensitising potential measured as structural alerts

Other effects / acceptance of results:

The predicted skin sensitisation potential of the main constituents of Castor oil, ester with glycerol as protein binding potential was modelled in the OECD QSAR Toolbox v4.0. The components fall within the model applicability domain for the database OASIS v1.4. No alerts were found. Therefore, the main constituents of Castor oil, ester with glycerol are not expected to have a skin sensitising potential and therefore the test substance Castor oil, ester with glycerol is not expected to have skin sensitising potential.

Interpretation of results:

study cannot be used for classification

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Analogue justification

No data on the skin sensitisation of Castor oil, ester with glycerol (CAS 68459-67-6) are available. The assessment was therefore based on QSAR modelling and studies conducted with analogue (source) substances as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. 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 analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

Skin sensitisation

QSAR predictions 

CAS 68459-67-6

The potential for the mono- and diester of Castor oil, ester with glycerol to exhibit skin sensitising properties was predicted in the QSAR OECD toolbox (WoE, 2017). The results for these components are considered to be representative for the target substance. Based on the results predicted with the OECD toolbox, there was no alert for skin sensitisation potential in the database OASIS v1.4, as estimated based on protein binding potential.

In vivo

CAS 555-43-1 (source substance)

Glycerol tristearate was investigated in male and female guinea pigs in a GLP-conform Buehler test according to OECD 406 (WoE, 1998). In a preliminary skin irritation test with 3 female animals, test substance formulations of 10, 20, 30 and 50% in petrolatum were topically applied to the flank under occlusive conditions for 6 h in order to establish a non-irritant concentration for induction. The concentration for challenge exposure was determined in a further 4-week test on 3 animals using the same concentrations. The maximum non-irritant concentration of 50% was used for topical application in the induction and challenge phase of the main assay. In the induction phase, the test substance at concentrations of 50% in petrolatum was applied to the clipped skin of the left flank of 20 animals using an occlusive dressing. During induction, three consecutive topical applications for a period of 6 h each were performed at intervals of 7 days. A control group of 10 animals was treated with the vehicle. For challenge exposure on Day 28, the test substance at 50% concentration in petrolatum and the vehicle only was applied for 6 h to the clipped skin of the posterior and anterior right flank of all animals, respectively. Skin reactions were evaluated 24 and 48 after application. None of the treated animals of the test and control group showed symptoms of dermal irritation after challenge treatment. No test substance-related systemic effects and no effects on body weights were observed in the test or control animals. The positive control substanceα-hexyl cinnamic aldehyde showed the expected results thereby confirming the reliability of the study. Based on these results and the experimental conditions chosen, the test substance had no skin sensitising effect in guinea pigs.

 

CAS 736150-63-3 (source substance)

The skin sensitising potential of Glycerides, castor-oil.mono, hydrogenated, acetates was investigated in a Local Lymph Node Assay (LLNA) in mice performed according to OECD guideline 429 and in compliance with GLP (WoE, 2007). In this study, 5 female CBA7CaOlaHsd mice per test group were treated with test substance at 10, 20 and 50% (w/v) in dimethyl sulfoxide or vehicle alone, respectively. The test substance formulations or the vehicle were applied topically to the dorsal surface of each ear lobe (25 µL/ear) for three consecutive days. Different batches (A-D) were used for treatment with the test substance at the respective concentrations. Five days after the first topical application, animals were sacrificed and the cell proliferation of pooled lymph nodes from individual animals was measured by incorporation of ³H-methyl thymidine and expressed as the amount of radioactive disintegration per minute (DPM). For batch A, the mean DPM/lymph node for each test group was 1528.3, 2088.3 and 3468.7 at concentrations of 10, 20 and 50% of the test substance, respectively. At 50% concentration of Batch A, a statistically significant difference (p ≤ 0.05) in DPM per lymph node was observed compared to control (DPM/node = 2027.9). Based on these results, stimulation indices of 0.75, 1.03 and 1.71 were calculated for the treatment concentrations of 10, 20 and 50%, respectively. The animals treated with the mid and the highest concentration (25% and 50%) of Batch A showed reddening of the ear skin from the second day of treatment. In summary, stimulation indices at of maximal 0.97 in the 10% groups, 1.53 in the 25% groups and 1.86 in the 50% groups were observed after treatment with the batches A-D of the test substance. Based on this data, no EC3 values of the test groups could be calculated, since none of the tested concentrations induced a stimulation index greater than 3. The positive control substance (25% hexyl cinnamic aldehyde in acetone:olive oil (4+1)) induced positive reactions in 2/5 animals (40%) and thus confirmed the sensitivity and reliability of the experimental technique. Under the above mentioned conditions, the test substance was not found to be a sensitiser in the LLNA.

Conclusion

A weight-of-evidence approach was applied to assess the skin sensitising potential of the target substance Castor oil, ester with glycerol. The OECD QSAR Toolbox did not predict protein binding indicative for sensitising properties of the mono- and diester of the target substance. In vivo studies (Buehler test and LLNA) performed with 2 source substances all gave negative results. Therefore, Castor oil, ester with glycerol is not expected to be skin sensitising. 

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” 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 analogue concept is applied to Castor oil, ester with glycerol, data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the analogue read-across approach, the available data on skin sensitisation do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.