Androst-4-ene-3,17-dione

Carcinogenicity

Description of key information

Results of 2-year carcinogenicity studies in rats and mice published within the scope of NTP (NIH, US) assessment [NTP Technical Report 560, NIH Publication No. 10-5901, September 2010]:
Oral, 2 years (Rat-Wistar, GLP, doses 0 / 10 / 20 / 50 mg/kg, once daily, OECD TG451): equivocal evidence of carcinogenic activity
Oral, 2 years (Mouse-B6C3F1, GLP, doses 0 / 10 / 20 / 50 mg/kg, once daily, OECD TG451): clear evidence of carcinogenic activity
Steroid hormones in general can promote the growth of specific hormone dependent tissues and tumors.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: NTP laboratory health and safety guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

1981

Principles of method if other than guideline:

Cited from report: "The NTP conducts its studies in compliance with its laboratory health and safety guidelines and FDA Good Laboratory Practice Regulations and must meet or exceed all applicable federal, state, and local health and safety regulations. Animal care and use are in accordance with the Public Health Service Policy on Humane Care and Use of Animals. Studies are subjected to retrospective quality assurance audits before being presented for public review."

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): Androstendione
- Lot/batch No.: H408
- Purity: > 98 %
- Stability under test conditions: The stability of the substance in the formulation was analytically verified for at least 35 days (sealed amber glass bottles; 5 °C).

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: F344/N
- Source: Taconic Farms, Inc. (Germantown, NY, USA)
- Age at study initiation: 5 to 6 weeks old
- Weight at study initiation (mean): males 121 g, females 103 g
- Housing: in groups of three (males) or five (females) per cage (solid bottom polycarbonate cages)
- Diet and Water: ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approx. 22 +/- 1.6 (72 +/- 3 °F)
- Humidity (%): 50 +/- 15
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

Administration volume: 5 mL/kg

VEHICLE: 0.5 % aqueous methylcellulose

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were analysed every 2 to 3 months during the 2-year study; animal room samples were also analysed. The analytical method used was HPLC.

Duration of treatment / exposure:

104 to 105 weeks

Frequency of treatment:

once daily, 5 days/week

Post exposure period:

no, animals were sacrificed on the day after the last dosing

Remarks:

Doses / Concentrations:
10, 20, and 50 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: For dose selection results from previously conducted 2-week and 3-month studies were used.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, all animals
- Time schedule: observed twice daily; clinical findings were recorded every 4 weeks

BODY WEIGHT: Yes, all animals
- Time schedule for examinations: Animals were weighed on day 1, day 4 (males), day 5 (females), weekly for 13 weeks, monthly thereafter, and at the end of the studies.

CLINICAL PATHOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, necropsies were performed on all animals

HISTOPATHOLOGY: Yes, complete histopathology was performed on all animals
In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone, brain, clitoral gland, esophagus, eye, harderian gland, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, spleen, stomach (forestomach and glandular), testis with epididymis, thymus, thyroid gland, trachea, urinary bladder, and uterus.

Statistics:

Survival Analysis: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Statistical analyses for possible dose-related effects on survival used Cox¿s (1972) method for testing two groups for equality and Tarone¿s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.
Calculation of Incidences were performed: Incidences of neoplasms or nonneoplastic lesions as the numbers of animals bearing such lesions at a specific anatomic site and the numbers of animals with that site examined microscopically.
Analysis of Neoplasm and Nonneoplastic Lesion Incidences: Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) to assess neoplasm and nonneoplastic lesion prevalence. Tests of significance included pairwise comparisons of each dosed group with controls and a test for an overall dose-related trend. Continuity-corrected Poly-3 tests were used in the analysis of lesion incidence, and reported P values are one sided.
Analysis of Continuous Variables: Two approaches were employed to assess the significance of pairwise comparisons between dosed and control groups in the analysis of continuous variables. 1) Parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). 2) Nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964).

Clinical signs:

no effects observed

Description (incidence):

Survival of 10 mg/kg males was significantly greater than that of the vehicle controls.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of 20 and 50 mg/kg females were greater than those of the vehicle controls after weeks 17 and 9, respectively.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The incidences of mononuclear cell leukemia were significantly increased in 20 and 50 mg/kg females and significantly decreased in 20 and 50 mg/kg males.
Incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) were significantly increased in 20 mg/kg males.
The incidence of testicular interstitial cell adenoma (including bilateral) was significantly decreased in 50 mg/kg males. In females, the incidences of mammary gland fibroadenoma were significantly decreased in the 20 and 50 mg/kg groups, the incidences of mammary gland hyperplasia were significantly decreased in all dosed groups, and the incidences of mammary gland cyst were significantly decreased in the 10 and 50 mg/kg groups.
In the liver of males, the incidences of basophilic focus in all dosed groups, the incidence of clear cell focus in the 20 mg/kg group, and the incidence of eosinophilic focus in the 50 mg/kg group were significantly increased.
The incidences of pancreatic islet hyperplasia and atrophy of the exocrine pancreas were significantly increased in 50 mg/kg females.
The incidence of lymphoid follicular hyperplasia of the spleen was significantly increased in 50 mg/kg females.
In the thyroid gland of males, the incidences of C-cell hyperplasia occurred with a negative trend and the incidence in the 50 mg/kg group was significantly decreased.
In female rats, the incidences of focal hypertrophy of the adrenal cortex occurred with a negative trend and the incidence in the 50 mg/kg group was significantly decreased.

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Cited from NTP report:

"Under the conditions of these 2-year gavage studies, there was equivocal evidence of carcinogenic activity of androstenedione in male F344/N rats based on increased incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined). There was equivocal evidence of carcinogenic activity of androstenedione in female F344/N rats based on increased incidences of mononuclear cell leukemia."

 

Conclusions:

equivocal evidence of carcinogenic activity

Executive summary:

Cited from NTP report:

"Groups of 50 male and 50 female rats were administered 0, 10, 20, or 50 mg androstenedione/kg body weight in a 0.5% aqueous methylcellulose solution by gavage, 5 days per week for at least 104 weeks. Survival of 10 mg/kg males was significantly greater than that of the vehicle controls. The mean body weights of 20 and 50 mg/kg females were greater than those of the vehicle controls after weeks 17 and 9, respectively."

"Under the conditions of these 2-year gavage studies, there was equivocal evidence of carcinogenic activity of androstenedione in male F344/N rats based on increased incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined). There was equivocal

evidence of carcinogenic activity of androstenedione in female F344/N rats based on increased incidences of mononuclear cell leukemia."

Androstenedione administration caused increased incidences in nonneoplastic lesions of the liver in male and female rats; pancreatic islets and exocrine pancreas of female rats. Decreases in the incidences of testicular interstitial cell adenoma in male rats, and mammary gland fibroadenoma, cysts, and hyperplasia in female rats were considered related to androstenedione administration.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: NTP laboratory health and safety guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

1981

Principles of method if other than guideline:

Cited from report: "The NTP conducts its studies in compliance with its laboratory health and safety guidelines and FDA Good Laboratory Practice Regulations and must meet or exceed all applicable federal, state, and local health and safety regulations. Animal care and use are in accordance with the Public Health Service Policy on Humane Care and Use of Animals. Studies are subjected to retrospective quality assurance audits before being presented for public review."

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): Androstendione
- Lot/batch No.: H408
- Purity: > 98 %
- Stability under test conditions: The stability of the substance in the formulation was analytically verified for at least 35 days (sealed amber glass bottles; 5 °C).

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: B6C3F1
- Source: Taconic Farms, Inc. (Germantown, NY, USA)
- Age at study initiation: 5 to 6 weeks old
- Weight at study initiation (mean): males 23.3 g, females 19.1 g
- Housing: singly (males) or in groups of five (females) per cage (solid bottom polycarbonate cages)
- Diet and Water: ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approx. 22 +/- 1.6 (72 +/- 3 °F)
- Humidity (%): 50 +/- 15
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

Administration volume: 10 mL/kg

VEHICLE: 0.5 % aqueous methylcellulose

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were analysed every 2 to 3 months during the 2-year studies; animal room samples were also analysed. The analytical method used was HPLC.

Duration of treatment / exposure:

104 to 106 weeks

Frequency of treatment:

once daily, 5 days/week

Post exposure period:

no, animals were sacrificed on the day after the last dosing

Remarks:

Doses / Concentrations:
10, 20, and 50 mg/kg bw
Basis:
other: males; actual ingested

Remarks:

Doses / Concentrations:
2, 10, 50 mg/kg bw
Basis:
other: females; actual ingested

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: For dose selection results from previously conducted 2-week and 3-month studies were used. The lower dose group of 2 mg/kg in females was selected due to suspected ovarian atrophy observed in the 3-month study; however, this finding was not confirmed upon reexamination.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, all animals
- Time schedule: observed twice daily; clinical findings were recorded every 4 weeks

BODY WEIGHT: Yes, all animals
- Time schedule for examinations: animals were weighed on day 1, day 4 (males), day 5 (females), weekly for 13 weeks, monthly thereafter, and at the end of the studies.

CLINICAL PATHOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, complete necropsies were performed on all rats

HISTOPATHOLOGY: Yes, complete histopathology was performed on all animals
In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone, brain, clitoral gland, esophagus, eye, gallbladder, harderian gland, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, spleen, stomach (forestomach and glandular), testis with epididymis, thymus, thyroid gland, trachea, urinary bladder, and uterus.

Statistics:

Survival Analysis: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Statistical analyses for possible dose-related effects on survival used Cox¿s (1972) method for testing two groups for equality and Tarone¿s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.
Calculation of Incidences were performed: Incidences of neoplasms or nonneoplastic lesions as the numbers of animals bearing such lesions at a specific anatomic site and the numbers of animals with that site examined microscopically.
Analysis of Neoplasm and Nonneoplastic Lesion Incidences: Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) to assess neoplasm and nonneoplastic lesion prevalence. Tests of significance included pairwise comparisons of each dosed group with controls and a test for an overall dose-related trend. Continuity-corrected Poly-3 tests were used in the analysis of lesion incidence, and reported P values are one sided.
Analysis of Continuous Variables: Two approaches were employed to assess the significance of pairwise comparisons between dosed and control groups in the analysis of continuous variables. 1) Parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). 2) Nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964).

Clinical signs:

no effects observed

Description (incidence):

Survival of dosed groups was similar to that of the vehicle control groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of 10 and 50 mg/kg females were generally less than those of the vehicle controls after weeks 81 and 17, respectively.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The incidences of hepatocellular adenoma in males and females were significantly increased in the 50 mg/kg groups. In females, the incidences of hepatocellular carcinoma were significantly increased in all dosed groups. Incidences of hepatocellular adenoma or carcinoma (combined) in males and females were significantly increased in the 50 mg/kg groups. Incidences of hepatoblastoma were marginally increased in dosed males. Incidences of multiple hepatocellular adenomas and carcinomas were significantly increased in 10 and 50 mg/kg males, and there was an increased incidence of multiple hepatocellular adenomas in 50 mg/kg females. The incidence of eosinophilic focus was significantly increased in 50 mg/kg males, and the incidences of mixed cell focus and cytoplasmic vacuolization were significantly increased in 50 mg/kg females.
There was a marginally increased incidence of pancreatic islet adenoma in 50 mg/kg males and in 10 and 50 mg/kg females, with an earlier day of first incidence in males. The incidences of clitoral gland hyperplasia and clitoral gland duct dilatation were significantly increased in 10 and 50 mg/kg females. The incidence of glomerular metaplasia of the kidney was significantly increased in 50 mg/kg females, and the incidences of cytoplasmic alteration of the submandibular salivary gland were significantly increased in all dosed female groups. The increased incidences of cytoplasmic alteration of the submandibular salivary gland and glomerular metaplasia of the kidney in female mice indicated a masculinizing effect from androstenedione treatment.
In 50 mg/kg females, the incidence of malignant lymphoma was significantly decreased.

The incidences of bone marrow hyperplasia were significantly increased in 10 and 50 mg/kg males, and the severities were increased in all dosed male groups.
The incidence of atrophy of the thymus was significantly increased in 20 mg/kg males. However, the biological significance of these findings is uncertain.

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Cited from NTP report:

"There was clear evidence of carcinogenic activity of androstenedione in male B6C3F1 mice based on increased incidences of multiple hepatocellular adenoma and hepatocellular carcinoma and increased incidence of hepatoblastoma. There was clear evidence of carcinogenic activity of androstenedione in female B6C3F1 mice based on increased incidences of hepatocellular adenoma and hepatocellular carcinoma. Increased incidences of pancreatic islet adenoma in male and female mice were also considered chemical related."

Executive summary:

Cited from NTP report:

"Groups of 50 male and 50 female mice were administered 0, 2 (females only), 10, 20 (males only), or 50 mg androstenedione/kg body weight in a 0.5% aqueous methylcellulose solution by gavage, 5 days per week for at least 104 weeks. Survival of dosed groups was similar to that of the vehicle control groups. Mean body weights of 10 and 50 mg/kg females were generally less than those of the vehicle controls after weeks 81 and 17, respectively."

"There was clear evidence of carcinogenic activity of androstenedione in male B6C3F1 mice based on increased incidences of multiple hepatocellular adenoma and hepatocellular carcinoma and increased incidence of hepatoblastoma. There was clear evidence of carcinogenic activity of androstenedione in female B6C3F1 mice based on increased incidences of hepatocellular adenoma and hepatocellular carcinoma. Increased incidences of pancreatic islet adenoma in male and female mice were also considered chemical related."

Androstenedione administration caused increased incidences in nonneoplastic lesions of clitoral gland, kidney, and submandibular salivary gland of female mice. Decreases in the incidences of malignant lymphoma in female mice were considered related to androstenedione administration.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Study duration:

chronic

Species:

mouse

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification on carcinogenicity warranted as Carc. 2 (H351: Suspected of causing cancer) according to Regulation (EC) 1272/2008.

 

Published studies on carcinogenicity, conducted within the scope of NTP (NIH, US) assessment, revealed equivocal evidence of carcinogenic activity in rats and clear evidence of carcinogenic activity in mice based on increased incidences of multiple hepatocellular adenoma and carcinoma and increased incidence of hepatoblastoma as well as increased incidence of pancreatic islet adenoma. However, for classification of androstendione it has to be taken into account that steroids hormones in general can promote the growth of specific hormone dependent tissues and tumors.

 

The classification is in accordance with German legislation for classification of androgenic steroids. (See argumentation for the assessment of steroid hormones, Technical Rule for Hazardous substances 905; elaborated by the German Committee on Hazardous Substances (AGS) and published by the German Federal Ministry of Labour and Social Affairs, version of 2008/2005/1999, only available in German, http://www.baua.de/de/Themen-von-A-Z/Gefahrstoffe/TRGS/Begruendungen-905-906.html).

Additional information

Results of two-year carcinogenicity studies on rats and mice were published within the scope of National Toxicology Program (NIH, US) assessment [NTP Technical Report 560, NIH Publication No. 10-5901, September 2010]:

 

In these studies androstendione was administered by gavage to male and female rats and male mice at dosages of 0, 10, 20, or 50 mg/kg body weight each day and to female mice at dosages of 0, 2,10, or 50 mg/kg body weight for two years. At the end of the study tissues from more than 40 sites were examined for every animal.

 

Cited from NTP report: "Under the conditions of these 2-year gavage studies, there was equivocal evidence of carcinogenic activity of androstenedione in male F344/N rats based on increased incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined). There was equivocal evidence of carcinogenic activity of androstenedione in female F344/N rats based on increased incidences of mononuclear cell leukemia. There was clear evidence of carcinogenic activity of androstenedione in male B6C3F1 mice based on increased incidences of multiple hepatocellular adenoma and hepatocellular carcinoma and increased incidence of hepatoblastoma. There was clear evidence of carcinogenic activity of androstenedione in female B6C3F1 mice based on increased incidences of hepatocellular adenoma and hepatocellular carcinoma. Increased incidences of pancreatic islet adenoma in male and female mice were also considered chemical related.
Androstenedione administration caused increased incidences in nonneoplastic lesions of the liver in male and female rats and mice; pancreatic islets and exocrine pancreas of female rats; and clitoral gland, kidney, and submandibular salivary gland of female mice.
Decreases in the incidences of testicular interstitial cell adenoma in male rats, mammary gland fibroadenoma, cysts, and hyperplasia in female rats, and malignant lymphoma in female mic were considered related to androstenedione administration."

 

However, for the assessment of carcinogenicity it has to be taken into account that steroid hormones in general can promote the growth of specific hormone dependent tissues and tumors.

Overall, androstendione caused liver cancer (variety of liver tumors, including adenomas, carcinomas and hepatoblastomas) and pancreatic islet cancer (adenomas) in male and female mice. The occurrence of lung tumors (a few adenomas and one carcinoma) in male rats and mononuclear cell leukemia in female rats may have been related to androstendione exposure. Increases in nonneoplastic lesions of the pancreas in female rats and of the clitoral gland, kidney, and salivary gland in female mice were attributed to androstendione exposure. Androstendione treatment reduced the incidence of neoplasms in several tissues that are well known endocrine targets (decreases in the incidences of testicular interstitial cell adenoma in male rats, mammary gland fibroadenoma, cysts, and hyperplasia in female rats, and malignant lymphoma in female mice).

 

For an assessment of steroid hormones see also the argumentation for steroid hormones related to the Technical Rule for Hazardous Substances 905, which was elaborated by the German Committee on Hazardous Substances (AGS) and published by the German Federal Ministry of Labour and Social Affairs (version of 2008/2005/1999, http://www.baua.de/de/Themen-von-A-Z/Gefahrstoffe/TRGS/Begruendungen-905-906.html, only available in German). In this argumentation 73 steroid hormones or precursors of steroid hormones were each allocated to one of seven hormone classes based on their predominant pharmacological activity (i.e. androgenic, mild androgenic, anabolic, estrogenic, gestagenic, mild gestagenic or glucocorticoide), and recommendations for their classification were elaborated.

Further supporting information is available:

A population-based case-control study was conducted examining hormone levels at birth among 91 infants (0–4 years) and 276 adolescents (15–19 years) diagnosed with TGCT, and 344 matched controls. Estrogen and androgen levels were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) from archived newborn dried blood spots. Logistic regression models were used to estimate the association between each hormone level and TGCT risk. Higher levels of neonatal androgens were associated with increased risk of TGCT among adolescents, suggesting that early life hormone levels are related to the later development of TGCT (Morimoto, 2018).

 

In a further study, short-term hepatic effects of androstenedione (A4) were compared to reference agonists of the estrogen receptor (ethinyl estradiol, EE) and glucocorticoid receptor (prednisone, PRED). Male and female B6C3F1 mice were exposed for 7 or 28 days to A4, EE, or PRED. The results of this study suggest that A4 induces subtle age-related effects on STAT5b signaling that may contribute to the higher risk of liver cancer in males compared to females (Rooney, 2017).

 

Further studies are cited in RTECS database (April 2013):

After subcutaeous application to mice over 9 weeks the animals developed tumours at the application site (carcinogenic by RTECS criteria); TDLo: 750 mg/kg/9W-I [Journal of the National Cancer Institute. (Washington, DC) V.1-60, 1940-78. For publisher information, see JJIND8. v. 19, p. 977, 1957 (JNCIAM)]