N,N-dimethyl-alkyl-1-amines, reaction products with alkali hydroxide and chloroacetic acid

Endpoint summary

Description of key information

OECD 408 (Repeated Dose 90-Day Oral): NOAELrat = 145 mg/kg bw/day a.i.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Comparable to guideline study. Guideline not explicitly stated in report, urinalysis not performed, no detailed clinical observations of the animals.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Guideline not stated, urinalysis not performed, no detailed clinical observations of the animals

GLP compliance:

yes

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 Wiga, Sulzfeld
- Age at study initiation: approx. 5 weeks (4 weeks at arrival)
- Weight at study initiation: males 51-65 g, females 52-72 g (at arrival)
- Fasting period before study: no data
- Housing: 2-3/cage
- Diet (e.g. ad libitum): pelleted, sterilized, nitrosamine-reduced maintenance diet Altromin 1324 DK, Altromin GmbH, Lage, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-25
- Humidity (%): 36-58
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Daily preparation, shortly before application.


VEHICLE
- Concentration in vehicle: 12.5, 25.0, 50.0 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg bw

Application volumes were adjusted weekly to compensate for changing body weights. Therefore, doses were kept constant over the whole experimental period.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

90 days (68 respective 69 applications, dependent on necropsy date)

Frequency of treatment:

once daily, 5 days/week

Remarks:

Doses / Concentrations:
0, 125, 250, 500 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: according to already established toxicity data; doses were chosen to include a safety margin, determine a not cumulative-toxic dose, the toxicity range and reversibility of eventually occuring cumulative toxic effects.
- Rationale for selecting satellite groups: Satellite groups of 5 animals/sex/dose for the control and the high dose groups were included to investigate reversibility of possible compound-related effects.
- Post-exposure recovery period in satellite groups: 35 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily during acclimatisation and twice daily during application period.
- Cage side observations included: mortality, symptoms of intoxication.


DETAILED CLINICAL OBSERVATIONS: No.


BODY WEIGHT: Yes, body weight and weight gain.
- Time schedule for examinations: at arrival, once during acclimatisation, weekly during application period.


FOOD CONSUMPTION: yes, weekly, groupwise, except for dates of clinical investigations.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, mean food consumption was calculated for males and females of each dose group as g/rat/week.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No.


WATER CONSUMPTION: Yes, mean water consumption was calculated as ml/rat/week for males and females of each dose group.
- Time schedule for examinations: weekly, groupwise, except for dates of clinical investigations.


OPHTHALMOSCOPIC EXAMINATION: Yes, with split lamp after application of Mydriaticum Roche R (Hoffmann-La Roche, Grenzach-Wyhlen).
- Time schedule for examinations: at the day before necropsy.
- Dose groups that were examined: control and high dose group.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 6 weeks of application (interim examination) and at the end of the study.
- Anaesthetic used for blood collection: yes, not further specified
- Animals fasted: No data
- How many animals: all animals, from retroorbital plexus, in EDTA-coated tubes.
- Parameters examined: red blood cell count (RBC), hematocrit (HCT), mean corpuscular volume (MCV), hemoglobin (HGB), leukocyte count (WBC), platelet count (PLT), differential blood count: band leukocytes (Bands), segmented leukocytes (Seg), lymphocytes (Lymph), eosinophils (Eo), monocytes (Mono), basophils (Baso), myelocites (My), juveniles (Ju), diverse (Div)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 6 weeks of application (interim examination) and at the end of the study.
- Animals fasted: No data
- How many animals: all animals, from retroorbital plexus, in non-coated tubes; serum was obtained from twice centrifuged blood samples
- Parameters examined: urea, creatinin (CREA), sodium (Na), potassium (K), glucose, calcium (CALC), alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), bilirubin (BILIR), chloride (CHLO), protein (PROT), cholesterol (CHOL)


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, animals were sacrificed by an overdose of ether and cardially exsanguinated after opening of the chest. Subsequently the corpse was examined, the eyes were removed (fixation according to Davidson), the hypodermis prepared, the abdomen, chest and the cranial cavity opened, the corpse was examined. Organs were examined in situ and after removal, and partially they were directly fixed in 10 % neutralised formalin (Formol-Fixx-Concentrate) after weighing. Absolute and relative weights were determined for the following organs: brain, testes, heart, liver, spleen, adrenals, kidneys, thymus. Animals of the satellite groups were treated in the same way after 35 treatment-free days.
HISTOPATHOLOGY: Yes, after fixation histological slices of the organs were prepared and stained with Hematoxylin/Eosin. The following organs were histologically examined: Aorta thoracica, eye, colon, glandular stomach, intestine, brain, urinary bladder, skin, heart, testes, hypophysis, cerebellum, liver, trachea, lung, maxillary lymph node, mesenteric lymph node, spleen, epididymides, adrenals, peripheral nerves, kidney, ovary, pancreas, prostate, seminal vesicle, thyroid, salivary gland, esophagus, skeletal muscles, thymus, uterus, forestomach, tongue.

Statistics:

Significant differences of body weights among the experimental groups were determined by the t-test according to L. Sachs, Statistische Auswertungsmethoden, 3rd ed., p.11, 212, Springer Publishers, Berlin, 1971. Significant differences of clinico-chemical and hematological parameters were determined according to Dunnett's t-test. The Steel-test was used for determination of significant differences of organ weights among the experimental groups.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

not adverse

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

not adverse

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

local effects

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

local effects

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no substance-related clinical signs in the exposed groups.

One female of the high dose group demonstrated emaciation, hunched posture, reduced activity and laboured breathing due to gavage dosing (chronic trauma of esophagus, confirmed by histopathology). One female of the mid and the low dose group each showed non-substance-related alopecia.
There was no substance-related mortality. Two males and two females of the high dose group were killed or died due to non-substance-related reasons (due to misapplication or harmed during sampling).

BODY WEIGHT AND WEIGHT GAIN
Males of the high dose group showed a slight reduction of mean body weights from week 10 until end of the study. Low dose females demonstrated a slight reduction of mean body weights at the beginning of the study and in weeks 1 and 6, high dose females showed a slight reduction only in week 1. The body weights of all other animals were always comparable with those of the control groups.

FOOD CONSUMPTION
Mean food consumption of the treated groups was comparable with that of the control groups. Some variations were observed which were not treatment-related.

WATER CONSUMPTION
Mean water consumption of the treated males was increased in a dose-related fashion. The high dose females demonstrated an increased water consumption, as well. Mean water consumption of the low and mid dose females was comparable to the control group.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related changes of the eyes in the high dose group, compared to the control group. Animals of the low and mid dose group were not investigated.

HAEMATOLOGY
At interim examination males of the mid dose group demonstrated a slight increase of leukocytes, at the final examination the high dose females showed slightly increased segmented leukocytes.

CLINICAL CHEMISTRY
At interim examination the mid dose males demonstrated slightly increased urea levels.

ORGAN WEIGHTS
High dose males showed reduced absolute gonad and brain weights, high dose females showed reduced absolute kidney weights. Mid dose males demonstrated slightly increased relative heart weights, and mid dose females demonstrated slightly reduced relative kidney weights. No other changes in organ weights were observed, and the observed differences were not considered to be substance-related.

GROSS PATHOLOGY
The only substance-related findings were white centres and/or edema in the mucosa of the forestomach in 5/8 males and 5/8 females of the high dose group. After 35 treatment-free days the high dose animals were free of substance-related pathological changes. A red centre was found at the bottom of the stomach in one female. All other findings were considered to occur spontaneously and not to be treatment-related (discolouration of the thymus, enlargement of mandibular lymph nodes and accessory spleen, and accumulation of fluid in the uterus).

HISTOPATHOLOGY: NON-NEOPLASTIC
The only substance-related findings were irritative changes in the forestomach of the high dose group, demonstrated by papillomatous hyperplasia of the mucosa, high-grade inflammation and ulcer formation. 3 males and 4 females of the mid dose group demonstrated irritative changes in the forestomach ranging from slight to moderate up to ulcer formation. The forestomachs of the low dose group were without findings except a slight irritation of the mucosa in one animal. Irritation was considered as local effect due to gavage dosing, but the effect was more pronounced in the high dose group in a dose-dependent fashion.
Irritative changes of the forestomach were also observed in 2 males and 3 females of the satellite group after 35 days without treatment.

Other findings included calcification of the Arteria pulmonalis, germinal hyperplasia of mandibular lymph nodes, hydronephrosis, accumulation of fluid in the uteri, hyperplasia of the mamma. Occasionally periorbital edema of various degrees due to retroorbital blood sampling was observed. Germinal hyperplasia of various degrees was regularly observed in animals of all groups. Histiocytic focuses with and without phagocytic activity were observed in the liver in nearly all groups. These findings were considered to be related to a spontaneously occurring bacterial infection of unknown etiology.

The high dose female which had died in narcosis demonstrated high-grade hyperemia of the lung, the male and the female which had died due to application mistakes both demonstrated lesions in the forestomach, and the exophtalmus was confirmed in the male that had to be killed after retroorbital blood sampling by the finding of high-grade periorbital inflammation.

Key result

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: water consumption (systemic effect)

Key result

Dose descriptor:

LOEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: water consumption (systemic effect)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no systemic adverse effects were observed up to and including the highest dose level

Critical effects observed:

not specified

Conclusion:

The NOEL after subchronic treatment with the test substance was considered to be 250 mg/kg bw/d. At this dose level only local irritative effects related to gavage dosing were observed in the forestomachs of the animals.

At 500 mg/kg bw/d, which was considered as LOEL, additionally a slight systemic effect in the form of increased water consumption occurred. The local irritative effect observed in the mucosa of the forestomach was increased in a dose-related fashion compared to the mid dose group.

There were no adverse effects on reproductive organs reported.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

not adverse

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

not adverse

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

local effects

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

local effects

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no substance-related clinical signs in the exposed groups.

One female of the high dose group demonstrated emaciation, hunched posture, reduced activity and laboured breathing due to gavage dosing (chronic trauma of esophagus, confirmed by histopathology). One female of the mid and the low dose group each showed non-substance-related alopecia.
There was no substance-related mortality. Two males and two females of the high dose group were killed or died due to non-substance-related reasons (due to misapplication or harmed during sampling).

BODY WEIGHT AND WEIGHT GAIN
Males of the high dose group showed a slight reduction of mean body weights from week 10 until end of the study. Low dose females demonstrated a slight reduction of mean body weights at the beginning of the study and in weeks 1 and 6, high dose females showed a slight reduction only in week 1. The body weights of all other animals were always comparable with those of the control groups.

FOOD CONSUMPTION
Mean food consumption of the treated groups was comparable with that of the control groups. Some variations were observed which were not treatment-related.

WATER CONSUMPTION
Mean water consumption of the treated males was increased in a dose-related fashion. The high dose females demonstrated an increased water consumption, as well. Mean water consumption of the low and mid dose females was comparable to the control group.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related changes of the eyes in the high dose group, compared to the control group. Animals of the low and mid dose group were not investigated.

HAEMATOLOGY
At interim examination males of the mid dose group demonstrated a slight increase of leukocytes, at the final examination the high dose females showed slightly increased segmented leukocytes.

CLINICAL CHEMISTRY
At interim examination the mid dose males demonstrated slightly increased urea levels.

ORGAN WEIGHTS
High dose males showed reduced absolute gonad and brain weights, high dose females showed reduced absolute kidney weights. Mid dose males demonstrated slightly increased relative heart weights, and mid dose females demonstrated slightly reduced relative kidney weights. No other changes in organ weights were observed, and the observed differences were not considered to be substance-related.

GROSS PATHOLOGY
The only substance-related findings were white centres and/or edema in the mucosa of the forestomach in 5/8 males and 5/8 females of the high dose group. After 35 treatment-free days the high dose animals were free of substance-related pathological changes. A red centre was found at the bottom of the stomach in one female. All other findings were considered to occur spontaneously and not to be treatment-related (discolouration of the thymus, enlargement of mandibular lymph nodes and accessory spleen, and accumulation of fluid in the uterus).

HISTOPATHOLOGY: NON-NEOPLASTIC
The only substance-related findings were irritative changes in the forestomach of the high dose group, demonstrated by papillomatous hyperplasia of the mucosa, high-grade inflammation and ulcer formation. 3 males and 4 females of the mid dose group demonstrated irritative changes in the forestomach ranging from slight to moderate up to ulcer formation. The forestomachs of the low dose group were without findings except a slight irritation of the mucosa in one animal. Irritation was considered as local effect due to gavage dosing, but the effect was more pronounced in the high dose group in a dose-dependent fashion.
Irritative changes of the forestomach were also observed in 2 males and 3 females of the satellite group after 35 days without treatment.

Other findings included calcification of the Arteria pulmonalis, germinal hyperplasia of mandibular lymph nodes, hydronephrosis, accumulation of fluid in the uteri, hyperplasia of the mamma. Occasionally periorbital edema of various degrees due to retroorbital blood sampling was observed. Germinal hyperplasia of various degrees was regularly observed in animals of all groups. Histiocytic focuses with and without phagocytic activity were observed in the liver in nearly all groups. These findings were considered to be related to a spontaneously occurring bacterial infection of unknown etiology.

The high dose female which had died in narcosis demonstrated high-grade hyperemia of the lung, the male and the female which had died due to application mistakes both demonstrated lesions in the forestomach, and the exophtalmus was confirmed in the male that had to be killed after retroorbital blood sampling by the finding of high-grade periorbital inflammation.

Key result

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: water consumption (systemic effect)

Key result

Dose descriptor:

LOEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: water consumption (systemic effect)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no systemic adverse effects were observed up to and including the highest dose level

Critical effects observed:

not specified

Conclusions:

Conclusion:
The NOEL for Betaines, C12-14 (even numbered)-alkyldimethyl, potassium salt; aqueous commercial product(s) was estimated to be 250 mg/kg bw/d. At this dose level only local irritative effects related to gavage dosing were observed in the forestomachs of the animals.

At 500 mg/kg bw/d, which was considered as LOEL, additionally a slight systemic effect in the form of increased water consumption occurred. The local irritative effect observed in the mucosa of the forestomach was increased in a dose-related fashion compared to the mid dose group.

There were no adverse effects on reproductive organs reported.

Executive summary:

One 90-day oral repeated dose toxicity study is available from the structural analogue CAS 68424-94-2. The NOEL for Betaines, C12-14 (even numbered)-alkyldimethyl, potassium salt; aqueous commercial product(s) was estimated to be 250 mg/kg bw/day from this study. As explained in the justification for type of information, the differences in molecular structure between the target and the source are unlikely to lead to differences in the oral NOEL that are higher than the typical experimental error of the test method.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

145 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 2) and consistent studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on similar structural and physico-chemical properties (refer to endpoint discussion for further details).
The selected study is thus sufficient to fulfil the standard information requirements set
out in Annex VIII-IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No
1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There is no data available on the repeated dose toxicity of N,N-dimethyl-alkyl-1-amines, reaction products with alkali hydroxide and chloroacetic acid. However, there is reliable data for the structural related substances CAS 68424-94-2 and the reaction mass of CAS 683-10-3 and CAS 2601-33-4. The target substance and the source substances show pronounced structural and physico-chemical similarities. Therefore, read-across was performed based on an analogue approach. For a detailed justification of the analogue approach, please refer to section 13 of the technical dossier.

There is data available from a Repeated Dose 90-Day Oral Toxicity Study with CAS 68424-94-2 according to OECD guideline 408 in rats (Pittermann, 1993) and from a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD guideline 422 with a reaction mass containing both CAS 683-10-3 and CAS 2601-33-4 (Whitlow et al., 2009) with respective range-finder (Whitlow and Flade, 2008).

In the subchronic 90-day study (Pittermann, 1993) doses of 125, 250 and 500 mg/kg bw/day of a test material containing 29 - 33% CAS 68424-94-2 were administered to rats daily by oral gavage. In the course of the study no substance-related mortality occurred, and no substance-related clinical signs were observed in the exposed groups. The only signs of systemic toxicity were a dose-related increase in water consumption of the males and the high dose females, and a slight reduction of mean body weights of the high dose males from week 10 until the end of the study. All other parameters investigated showed no or only slight variations compared to the control group. Therefore, the mid-dose of 250 mg/kg bw/day was determined as NOEL, whereas the high-dose of 500 mg/kg bw/day was determined as LOEL. Considering the absence of other systemic effects like changes in clinical chemistry parameters, the LOEL can be regarded, at the same time, as NOAEL, as well. Converted to active ingredient this dose corresponds to 145 mg/kg bw/day, based on the assumption of 29% active ingredient in the test material. There were no adverse effects on reproductive organs reported in this study.

The combined repeated dose/reproductive toxicity study has been performed with a test substance consisting of 19.84% CAS 683-10-3 and 6.72% CAS 2601-33-4 (Whitlow et al., 2009). The test substance was applied to rats for 49 days by oral gavage. No systemic effects were observed in the low dose group, whereas signs of systemic toxicity like salivation, increased urea and histopathological findings in kidney and bladder were observed at the mid dose of 150 mg/kg bw/day. Therefore, the low dose of 50 mg/kg bw/day was determined as NOAEL, the corresponding LOAEL for systemic toxicity was 150 mg/kg bw/day. The corresponding range-finder (Whitlow and Flade, 2008) revealed a NOAEL of 100 mg/kg bw/day, the LOAEL of 300 mg/kg bw/day was characterised by signs of sedation, significantly reduced food consumption and body weight gain, very likely due to the fact that animals were sedated for most of the pre-pairing period. At 1000 mg/kg bw/day all animals died within 24 hours after the first treatment.

Although lower No Observed Adverse Effect Levels were demonstrated by the combined repeated dose/developmental toxicity screening test, the 90-day study (Pittermann, 1993) was chosen as key study, as for regulatory purposes the results of a subchronic study are required. Besides, the applied doses of all studies are within comparable ranges and the test material used for the subchronic study is well comparable to that of the combined repeated dose/developmental toxicity screening test. Therefore, the symptoms described in the latter would have also been expected in the subchronic study, especially in the high dose group, due to the longer study duration. However, histopathology in the study by Whitlow et al. demonstrated irritant effects in the stomach due to repeated gavage of an irritant test material, and in the kidney and bladder due to the excretion of an irritant test article or its metabolites in the urine. Therefore, it is reasonable to consider the effects described in this study, although adverse, as secondary to the observed inflammation reactions.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Hazard assessment is conducted by means of read-across from structural analogues. All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substances and overall overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details).

Justification for classification or non-classification

The available data on oral repeated dose toxicity of the analogue substances do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.