Reaction product of alkylarylsulphonic acid and cycloaliphatic amine

Endpoint summary

Description of key information

On AMP oral repeated dose studies are available in dogs (maximum concentrations in diet tested 2.8 and 62.5 mg/kg bw) and rats (90-day diet NOAEL 25 mg/kg bw). The effects seen in both species are in the liver which can be related to an effect on choline synthesis, but a clear species difference becomes apparent with the dog being more sensitive (lowest NOAEL 0.63 mg/kg bw). In a 2 -generation study with 4,4-dimethyl oxazolidine a parental NOAEL of 20 mg/kg bw was found (effects on liver cells).

For PTSA limited information is available from a 28-day toxicity study with a NOAEL of 500 mg/kg bw (no effects at the highest dose tested).

Aromatic sulphonic acids are almost completely ionized in watery environments even at low pH. In principle the salts of these acids get dissociated when in contact with water, so forming back to the acids. This is particularly relevant for studies that are conducted in water (e.g., ecotoxicity and biodegradation) as well as for mammalian toxicity studies where the relatively high acidity of the acid form has an immediate and harsh local effect, whereas the salt form provides an indication of potential systemic toxicity beyond the site of application or initial contact. Taking this into consideration and in view of the higher toxicity of 4,4 -dimethyl oxazolidine (and AMP), the NOAEL for oxazolidine is taken as starting point for the risk assessment.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5/19/1988 to 7/25/1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: meets generally accepted scientific standards, well-documented, and acceptable for assessment

Remarks:

maximum dose level low and without effects

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

GLP compliance:

yes (incl. QA statement)

Remarks:

contains an audit certificate

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

Twenty nine male and 29 female beagle dogs were received from Laboratory Research Enterprises, Kalamazoo, MI, and 25 of the healthiest of each sex were used for study. The animals were selected based on criteria of the US FDA (1982). The dogs were 4 months of age at arrival to the testing laboratory. Animals were identified by an assigned identification number on a collar, housed singly for an acclimation period, and were examined by a laboratory veterinarian for general health status. Animals were stratified by body weight, and assigned randomly to their treatment groups.

Route of administration:

oral: feed

Vehicle:

other: test material was dissolved in ethanol and then mixed into the feed

Details on oral exposure:

Animals were dosed via the diet, which was tested by the supplier (Purina) for contaminants. The test article was incorporated into the diet on a weight/weight basis using a premix. Fresh food was prepared with the test article weekly. The animals were offered 400g of the diets daily. Any uneaten food was weighed and recorded.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

1 year

Frequency of treatment:

continuous

Remarks:

Doses / Concentrations:0, 1.1, 11, 110 ppmBasis:nominal in diet

Remarks:

Doses / Concentrations:0, 0.031, 0.31, 2.8 mg/kg bw/dayBasis:nominal in diet

No. of animals per sex per dose:

6/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Animals were dosed via the diet, which was tested by the supplier (Purina) for contaminants. The test article was incorporated into the diet on a weight/weight basis using a premix. Fresh food was prepared with the test article weekly. The animals were offered 400g of the diets daily. Any uneaten food was weighed and recorded. Drinking water was provided throughout the study ad libitum. Six males and six females were assigned to each of 4 dose groups (0, 1.1, 11, 110 ppm).Daily observations were made of the general appearance, behavior, the presence of any signs of toxicity or pharmacologic effects and mortality. Body weights were recorded prior to dosing, and weekly thereafter. The fasting terminal body weight was also recorded. While weighing, the animals were examined for lesions or other signs of toxicity. Each animal had an ophthalmoscopic examination once pre-study, weeks 1, month 3,6,9,12 by a veterinarian.Blood samples were obtained pre-study, and at 3, 6, 9, 12 months via venipuncture of fasted animals. The following parameters were recorded: hematocrit, hemoglobin, erythrocyte count, leukocyte count, platelet count, prothrombin time, MCH, MCHC, and MCV, Calcium, Phosphorous, Chloride, Sodium, Potassium, glucose, serum alkaline phosphatase, serum aspartate aminotransferase, serum analine aminotransferase, gamma glutamyl, transpeptidase, blood urea nitrogen, total protein, albumin, globulin, creatinine, bilirubin, and serum protein. Urine was examined for pH, specific gravity, volume and appearance, protein, glucose, acetone bodies, creatinine, and microscopic sediment.Post-mortem examinations included a complete gross examination of the external surfaces, all orifices, the cranial cavity, the external surface of the brain, the thoracic, pelvic, and abdominal cavities and their viscera, cervical tissues and organs, and the carcass. Two animals per sex per dose were sacrificed at 6 months, with the remainder at 12 months. Organ weights were recorded for the liver, kidneys, testes, thyroids, adrenals, and brain. Tissues that were removed and preserved are the same as current OECD guidelines

Positive control:

no

Observations and examinations performed and frequency:

Daily observations were made of the general appearance, behavior, the presence of any signs of toxicity or pharmacologic effects and mortality. Body weights were recorded prior to dosing, and weekly thereafter. The fasting terminal body weight was also recorded. While weighing, the animals were examined for lesions or other signs of toxicity. Each animal had an ophthalmoscopic examination once pre-study, weeks 1, month 3,6,9,12 by a veterinarian.

Sacrifice and pathology:

Blood samples were obtained pre-study, and at 3, 6, 9, 12 months via venipuncture of fasted animals. The following parameters were recorded: hematocrit, hemoglobin, erythrocyte count, leukocyte count, platelet count, prothrombin time, MCH, MCHC, and MCV, Calcium, Phosphorous, Chloride, Sodium, Potassium, glucose, serum alkaline phosphatase, serum aspartate aminotransferase, serum analine aminotransferase, gamma glutamyl, transpeptidase, blood urea nitrogen, total protein, albumin, globulin, creatinine, bilirubin, and serum protein. Urine was examined for pH, specific gravity, volume and appearance, protein, glucose, acetone bodies, creatinine, and microscopic sediment.Post-mortem examinations included a complete gross examination of the external surfaces, all orifices, the cranial cavity, the external surface of the brain, the thoracic, pelvic, and abdominal cavities and their viscera, cervical tissues and organs, and the carcass. Two animals per sex per dose were sacrificed at 6 months, with the remainder at 12 months. Organ weights were recorded for the liver, kidneys, testes, thyroids, adrenals, and brain. Tissues that were removed and preserved are the same as current OECD guidelines

Statistics:

yes

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):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Test material intake is estimated based on week 36 body weights and food consumption.Males estimated (mg/kg/day)Control 01.1ppm .03111ppm 0.31110ppm 2.98Females estimated (mg/kg/day)Control 01.1ppm .02911ppm 0.31110ppm 2.55There were no general in-life observations made for any treated or control animal. Likewise, there were no ophthalmoscopic observations noted for any animal. Neither an ANOVA or a Kruskal-Wallis non-parametric ANOVA revealed any statistical significance in food consumption patterns for any dose level in either sex. There was no statistically significant differences in body weights for any of the animals throughout the study. Clinical chemistry findings for males revealed a slight decrease in the low and mid-dose groups' albumin-globulin ratio. Since there was no dose-response (the high dose group was not different than the control), it was judged to be a spurious finding and not treatment-related. High-dose males at 9 and 12 months showed differences in serum albumin via serum electrophoresis, but was not corroborated by actual clinical chemistry measurements, and was not considered by the authors to be biologically-significant. Throughout the study, findings for females included differences in serum sodium, serum ALT, and serum AST, but were transient, did not appear dose-related, and were judged by the authors to be not related to the administration of AMP. There was no effect of AMP administration on hematology, and likewise the urinalysis revealed no treatment-related effects. There were no effects on organ weights or organ/body weight ratios that were attributed to test material administration by the authors.At necropsy, evaluation of the tissues revealed several isolated observations, none of which were attributed to the administration of the test material. There were no neoplastic observations in any of the dogs sacrificed at either 6 months or one year of administration in their diets.

Dose descriptor:

NOAEL

Effect level:

> 110 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Dose equivalent to 2.8 mg/kg bw/day based on average male and femal test material consumption

Critical effects observed:

not specified

Conclusions:

Based on the findings under these study conditions, there is no effect at any dose level on general appearance, behavior, body weight, food consumption, ophthalmoscopic exams, clinical chemistry, hematology, organ weights, or tissue histopathology. Based on the absence of statistically and biologically significant findings in dose-response patterns, the No-Observed Effect Level for AMP in the diets of Beagle dogs in greater than 110 ppm (>2.8 mg/kg bw).

Reference 2

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:

other: meets generally accepted scientific standards, well-documented, and acceptable for assessment

Remarks:

dose level intervals were not adequate

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

GLP compliance:

not specified

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

Study was run with Beagle dogs, 4/sex/dose. They were 4.5-5 months of age at arrival to the Bio/dynamics laboratory, and 5.5-6 months of age at the initiation of the study. Weight range at the start of treatment was 7.7-9.6kg for males (mean 8.7kg), and was 6.3-9.7 for females (mean 8.1kg). The dogs were immunized previously against distemper, hepatitis, leptospirosis, and rabies by the supplier. Fecal examinations were conducted on all dogs by the performing lab during the one month acclimation period. Baseline clinical laboratory tests were also performed. They were housed in elevated metal grid cages, and fed 400g of Purina Canine Meal Diet (#5007) presented for 4.5 hours. Fresh food was presented daily. Water was provided ad libitum by an automated water system. The dogs were maintained on a 12-hour photocycle, and temperature was monitored twice daily.Dogs were ranked by body weight and distributed into 4 groups of 4/sex so that body weights in each group were comparable. Groups were assigned to control and dose levels randomly. Each dog was assigned a unique identification number on an ear tag and on the animal's cage. In addition, each dog had an ear tatoo bearing the USDA number.

Route of administration:

oral: feed

Vehicle:

other: feed

Details on oral exposure:

The AMP was administered orally via the diet as AMP-95/HCl to 32 Beagle dogs (4/sex/dose group) at dose levels of 0, 25, 600, and 2500 ppm for a three month period. The control animals received an untreated diet.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Feed samples of each dose level were assayed for homogeneity, stability, and verification of dose concentration. Results were presented in a subsequent report from the performing laboratory, and not presented in this report.

Duration of treatment / exposure:

3 months

Frequency of treatment:

continuous

Remarks:

Doses / Concentrations:0, 25, 600, and 2500 ppm Basis:nominal in diet

Remarks:

Doses / Concentrations:0, 0.63, 15, 62.5 mg/kg bwBasis:nominal in diet

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Details on study design:

AMP was administered orally via the diet as AMP-95/HCl to 32 Beagle dogs (4/sex/dose group) at dose levels of 0, 25, 600, and 2500 ppm (0, 0.63, 15, 62.5 mg/kg bw) for a three month period. The control animals received an untreated diet. Body weights were measured pretest, weekly thereafter, and after fasting at termination; food consumption was measured daily and presented at weekly intervals. Feed samples of each dose level were assayed for homogeneity, stability, and verification of dose concentration. Results were presented in a subsequent report from the performing laboratory, and not presented in this report. Animals were observed for mortality and gross signs of toxicologic or pharmacologic effects twice daily, and dogs were given a detailed physical examination pre-test and weekly thereafter. Ophthalmoscopic examinations were performed pretest and at termination. Hematology, clinical chemistry, and urinalysis parameters were analyzed twice pre-test, and Month 1 and 3. Blood was obtained via jugular venipuncture. Urine was collected in stainless steel metabolism pans attached to each animal's permanent cage. Dogs were fasted overnight prior to blood collections, and were not dosed until after samples were collected. Hematology parameters evaluated included: hemoglobin, hematocrit, erythrocytes, platelets, clotting time, prothrombin time, and total and differential leukocytes. Clinical Chemistry parameters included: serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase, lactic acid dehydrogenase, blood urea nitrogen, fasting glucose, cholesterol, total protein, albumin, globulin, A/G ratio, total bilirubin, direct bilirubin, sodiom, potassium, chloride, and calcium. Urine was evaluated for the following: Gross appearance, specific gravity, pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen, and microscopic analysis.On days 96-99, animals were necropsied and tisses were preserved for weights (brain, ovaries, pituitary, thyroid, heart, adrenals, spleen, testes, kidneys, liver) and microscopic evaluation (the above plus aorta, bone, sternum, epididimus, esophagus, eye, colon, duodenum, ileum, lungs, mesenteric lymph node, mammary gland, sciatic nerve, pancreas, parathyroids, prostate, salivary gland, skeletal muscle, skin, spinal cord, stomach, thymus, thyroid, trachea, urinary bladder, and gross lesions). Statistical analyses were performed on data collected for body weight, food consumption, hematology, clinical chemistry, organ weights, and organ:body weight ratios. Mean values of all dose groups were compared to control at each time interval (where appropriate). Statistically significant differences from the controls are indicated.Species and strain of test animals, route and method of test substance administration, and dose levels were established by the sponsor. The study was performed at Bio/dynamics, INC.

Positive control:

no

Observations and examinations performed and frequency:

Body weights were measured pretest, weekly thereafter, and after fasting at termination; food consumption was measured daily and presented at weekly intervals. Feed samples of each dose level were assayed for homogeneity, stability, and verification of dose concentration. Results were presented in a subsequent report from the performing laboratory, and not presented in this report. Animals were observed for mortality and gross signs of toxicologic or pharmacologic effects twice daily, and dogs were given a detailed physical examination pre-test and weekly thereafter. Ophthalmoscopic examinations were performed pretest and at termination.

Sacrifice and pathology:

Hematology, clinical chemistry, and urinalysis parameters were analyzed twice pre-test, and Month 1 and 3. Blood was obtained via jugular venipuncture. Urine was collected in stainless steel metabolism pans attached to each animal's permanent cage. Dogs were fasted overnight prior to blood collections, and were not dosed until after samples were collected. Hematology parameters evaluated included: hemoglobin, hematocrit, erythrocytes, platelets, clotting time, prothrombin time, and total and differential leukocytes. Clinical Chemistry parameters included: serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase, lactic acid dehydrogenase, blood urea nitrogen, fasting glucose, cholesterol, total protein, albumin, globulin, A/G ratio, total bilirubin, direct bilirubin, sodiom, potassium, chloride, and calcium. Urine was evaluated for the following: Gross appearance, specific gravity, pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen, and microscopic analysis.On days 96-99, animals were necropsied and tisses were preserved for weights (brain, ovaries, pituitary, thyroid, heart, adrenals, spleen, testes, kidneys, liver) and microscopic evaluation (the above plus aorta, bone, sternum, epididimus, esophagus, eye, colon, duodenum, ileum, lungs, mesenteric lymph node, mammary gland, sciatic nerve, pancreas, parathyroids, prostate, salivary gland, skeletal muscle, skin, spinal cord, stomach, thymus, thyroid, trachea, urinary bladder, and gross lesions).

Statistics:

Statistical analyses were performed on data collected for body weight, food consumption, hematology, clinical chemistry, organ weights, and organ:body weight ratios. Mean values of all dose groups were compared to control at each time interval (where appropriate). Statistically significant differences from the controls are indicated.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortality:All animals survived the duration of the study.Physical ObservationsIncluded emesis, loose stool, alopecia, and slight penile or vaginal discharge. These findings occurred sporadically in the control and/or treated animals and are not considered by the authors to be treatment-related.OphthalmologyPretest and terminal examinations revealed no significant ocular changes through the study. There is no evidence of compound-induced ocular damage.Body Weight: No statistically-significant differences were found in any dose group when compared to animals of the same sex in the control group. There was a 10% mean body weight reduction noted in the high dose group relative to the control for males and females in latter phases of the study, suggesting to the authors a slight compound-related effect.Food ConsumptionThere was no treatment-related effect of the test material on food consumption at any dose level.HematologyPretest hematology values were within the normal physiological range for all animals. There were no statistically-significant differences between the control and treated animals at Month 1 testing. At Month 3, there was a trend toward reduced prothrombin time observed in the treated males and females with statistically significant differences noted in the top two dose levels for males only (p<0.01). In addition, a slight trend torward reduced hemoglobin concentrations was found in the treated females. Only the mean value for the high dose, which was slightly below the normal physiological range, was statistically significant (p<0.05). No other biologically significant differences were observed in any other hematology parameter evaluated.Clinical ChemistryPretest mean clinical chemistry values were within the normal physiological range for males and females. At both the Month 1 & 3 evaluations, statistically significant elevations in serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, and alkaline phosphatase activities occurred in the high dose males and females. The magnitude of the increases ranged from 2-4 fold for serum glutamic oxaloacetic transaminas, 12-28 fold for serum glutamic pyruvic transaminase, and 5-6 fold for alkaline phosphatase. These significant increases were limited to the high dose animals only, and were similar at months 1 & 3 for both sexes. Other findings included reduced mean cholesterol values in both sexes at the above intervals, while direct bilirubin concentrations were slightly higher in the treated animals. No other toxicologically significant differences occurred in the other clinical chemistry parameters evaluated. The above effects observed in the high dose animals suggest a high degree of hepatocellular damage in the high dose animals with no significant changes in the low or mid dose groups. The combination of increased serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase levels, both of which are present in high concentrations in hepatocytes, are indicative of hepatocellular necrosis. The increased alkaline phosphatase and direct bilirubin levels are suggestive of cholestasis and/or bile duct epithelial necrosis. It appears, therefore, that the liver is the promary target organ, based on the clinical chemistry data, when the test substance is administered via the diet.UrinalysisThere is no evidence of a compound-related effect on any of the urinalysis parameters studied.Organ Weight/ Organ:Body Weight RatiosA trend towards increased liver and/or liver/body weight rations was ovserved in both the mid and high dose male and female dogs. These differences were slight but tend to support the conclusion that the liver is the principle site of toxic action. Also, mean kidney weights and kidney/body weight ratios were increased in the high dose males, and in mid and high dose females. Other organ weight and organ/body weight differences were marginal and appear unrelated to the administration of the test material.PathologyTwo females and one male from the high dose group were found to have tan discoloration and mottling in liver upon gross postmortem evaluations. No other such findings were present in any of the other treated or control animals. Other gross findings occurred sporadically or were present in the treated and control animals to a similar degree. Microscopic findings included vacuolization, periportal cirrhosis, and bile duct hyperplasia in the liver sections in all high-dose males and females. Lipid deposition within the cellular vacuoles was confirmed by staining. The periportal cirrhosis was characterized by hepatocellular necrosis and fibrosis. These findings were more severe in the high-dose females as compared to the males. In addition, one low dose female had liver findings similar to the above, while 2 mid-dose males exhibited minimal fatty changes in the liver. Other microscopic findings occurred sporadically in the treated and/or control animals and were not related to the administration of the test material. The above microscopic findings correlate well with the clinical chemistry data, as both indicate hepatotoxicity induced by the test material in the high dose animals. The findings noted in the liver of the one low dose female, in the absense of altered clinical chemistry data, as well as the absence of similar microscopic findings in the mid dose group, are considered to be of uncertain significance (this may represent a response to treatment in an unusually sensitive animal, or may be a spontaneous finding)

Dose descriptor:

NOAEL

Effect level:

25 ppm

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

LOAEL

Effect level:

600 ppm

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

not specified

Conclusions:

The results of this study agree with those from a concurrently-run study (Bio/dynamics #80-2503), utilizing the same test material and similar dose levels in rats, which also indicated the liver is the principle target organ from dietary administration of the test material. The degree of changes in the present study clearly indicates that dogs are more sensitive to the hepatotoxic effects of the test material.

Reference 3

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:

other: see 'Remark'

Remarks:

Generally in line with the standard protocol. However there was a high incidence of viral infection in test animals (all groups). This was not considered to have affected the outcome of the study (viral infection of eyes only) but additional stress of the eye infection may have caused the animals to react more severely than they otherwise would.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

Appears to follow the guideline but not specifically stated in the report

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female Sprague-Dawley rats were received from Charles River Breeding Laboratories and were 4 weeks of age at arrival to the Bio/dynamics Lab. They were allowed to acclimate to the laboratory for a minimum of 14 days, and were housed individually in elevated stainless steel cages. They were fed Purina Certified Lab Chow (#5002) ad libitum, and fresh food was presented weekly. Water was provided ad libitum via an automated water system. Photocycle was 12 hours light per 24 hour cycle, and temperature was monitored twice daily. They were approximately 7.5 weeks of age at the start of treatment. Animals were given a pre-assignment physical examination and unsuitable animals were excluded from assignment to test groups. The animals were assigned randomly to groups (20/sex/dose), and were identified with an numeric metal ear tag.

Route of administration:

oral: feed

Vehicle:

other:

Details on oral exposure:

Appropriate amounts of test material were mixed with standard lab rodent diet weekly, to provide 0, 25, 150, 250, or 2500 ppm AMP to the animals. The animals received the test material continuously in the diet for 90-92 days prior to necropsy.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity of the test material in the feed was assayed by sampling all dose levels (from the top, middle, and bottom of the containers). Samples from all dose levels were assayed for stability in the feed at day 0, 3, 4, 7, 10, and 14. Concentration of the test material in the feed was analyzed for all dose levels on test weeks 1, 2, 3, 4, 6, 8, 12, and 13.

Duration of treatment / exposure:

90-92 days

Frequency of treatment:

continuous

Remarks:

Doses / Concentrations:0, 25, 150, 250, 2500 ppm Basis:nominal in diet

Remarks:

Doses / Concentrations:0, 0.25, 15, 25, or 250 mg/kg bwBasis:nominal in diet

No. of animals per sex per dose:

20/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Appropriate amounts of test material were mixed with standard lab rodent diet weekly, to provide 0, 25, 150, 250, or 2500 ppm to the animals. The animals received the test material continuously in the diet for 90-92 days prior to necropsy. Homogeneity of the test material in the feed was assayed by sampling all dose levels (from the top, middle, and bottom of the containers). Samples from all dose levels were assayed for stability in the feed at day 0, 3, 4, 7, 10, and 14. Concentration of the test material in the feed was analyzed for all dose levels on test weeks 1, 2, 3, 4, 6, 8, 12, and 13.Animals were observed twice daily for mortality and gross signs of toxicologic or pharmacologic effects. A detailed physical examination for signs of local or systemic toxicity, pharmacologic effects, and palpable masses was performed weekly. Ophthalmoscopic examinations were performed pretest and at the end of month 1 and 3. Body weights were recorded twice pretest, weekly during treatment, and terminally after fasting. Food consumption was recorded weekly beginning the week prior to study start, and test material was calculated from food consumption data based on nominal concentration of the test material in the feed.Blood was analyzed for the following parameters (pretest, month 1 & 3): hemoglobin, hematocrit, erythrocytes, platelets, total & differential leukocytes, erythrocyte morphology, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase, blood urea nitrogen, fasting glucose, total protein, albumin, globulin, A/G ratio, cholesterol, sodium, potassium, calcium, total bilirubin, and lactic acid dehydrogenase. Urine was analyzed at month 1 and 3, and gross appearance, specific gravity, pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen, and microscopic analysis were evaluated.Animals dying spontaneously received a gross postmortem exam. All others surviving to necropsy were euthanized under anesthesia, underwent a gross postmortem exam, and select tissues were weighed and/or preserved for examination. Organ weight was collected for adrenals, brain, gonads, heart, kidneys, liver, pituitary, and spleen and they were also preserved for examination. In addition, the following were preserved: aorta, blood smear, bone and bone marrow, epididymus, eye, cecum, ileum, duodenum, jejunum, lungs, mesenteric & mediastinal lymph nodes, mammary glands, sciatic nerve, pancreas, parathyroid, prostate, salivary gland, seminal vesicle, skeletal muscle, skin, spinal cord, stomach, thymus, thyroid, trachea, bladder, uterus, gross lesions, and tissue masses.Statistical analysis included body weight, food consumption, hematology, and clinical chemistry parameters, organ weights and organ:body weight ratios. Mean values of all dose groups were compared to control at each time interval where appropriate. Statistically significant differences from control are indicated in appendices.

Positive control:

no

Observations and examinations performed and frequency:

Animals were observed twice daily for mortality and gross signs of toxicologic or pharmacologic effects. A detailed physical examination for signs of local or systemic toxicity, pharmacologic effects, and palpable masses was performed weekly. Ophthalmoscopic examinations were performed pretest and at the end of month 1 and 3. Body weights were recorded twice pretest, weekly during treatment, and terminally after fasting. Food consumption was recorded weekly beginning the week prior to study start, and test material was calculated from food consumption data based on nominal concentration of the test material in the feed.

Sacrifice and pathology:

Blood was analyzed for the following parameters (pretest, month 1 & 3): hemoglobin, hematocrit, erythrocytes, platelets, total & differential leukocytes, erythrocyte morphology, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase, blood urea nitrogen, fasting glucose, total protein, albumin, globulin, A/G ratio, cholesterol, sodium, potassium, calcium, total bilirubin, and lactic acid dehydrogenase.Urine was analyzed at month 1 and 3, and gross appearance, specific gravity, pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen, and microscopic analysis were evaluated.Animals dying spontaneously received a gross postmortem exam. All others surviving to necropsy were euthanized under anesthesia, underwent a gross postmortem exam, and select tissues were weighed and/or preserved for examination. Organ weight was collected for adrenals, brain, gonads, heart, kidneys, liver, pituitary, and spleen and they were also preserved for examination. In addition, the following were preserved: aorta, blood smear, bone and bone marrow, epididymus, eye, cecum, ileum, duodenum, jejunum, lungs, mesenteric & mediastinal lymph nodes, mammary glands, sciatic nerve, pancreas, parathyroid, prostate, salivary gland, seminal vesicle, skeletal muscle, skin, spinal cord, stomach, thymus, thyroid, trachea, bladder, uterus, gross lesions, and tissue masses.

Statistics:

Statistical analysis included body weight, food consumption, hematology, and clinical chemistry parameters, organ weights and organ:body weight ratios. Mean values of all dose groups were compared to control at each time interval where appropriate. Statistically significant differences from control are indicated in appendices.

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):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Remark- Actual doses of test material in this study were calculated based on the bodyweight and feed consumption. The test material was AMP/HCL which thus contained approximately 50% AMP. Therefore the actual doses of AMP (approximate means) given were:Week 1Males: 0, 3, 17.5, 30, 300 mg/kg bw/dayFemales: 0, 3, 18, 32, 310 mg/kg bw/dayWeek 9 Males: 0, 1.6, 10, 16, 160 mg/kg bw/dayFemales: 0, 2, 12, 20, 210 mg/kg bw/dayAvearage dose over the course of the study:Males: 0, 2.4, 14, 23, 230 mg/kg bw/dayFemales: 0, 2.6, 15, 26, 260 mg/kg bw/dayMortalityAll animals survived the test material administration, with the exception of 2 females- one died following Month 1 blood collection, and the other was sacrificed on test day 18 due to poor food consumption, constipation, lethargy, and swollen abdomen. The condition was not considered due to the test material.Physical ObservationsAll observations noted were interpreted by the author to be related to a viral infection (sialdacryoadenitis) in all dose groups at approximately the same frequency, primarily in males except where noted, and included: alopecia, swollen salivary glands (both sexes), opacities, excessive lactimation, and chromodacryorrhea. Ophthalmoscopic ExaminationsPretest exams revealed no notable abnormalities. At months 1 & 3, a significant increase in a variety of lesions, which appeared to be infectious in origin, were noted. The observations were likely secondary to the sialodacryoadenitis infection which can lead to opacity and other ocular lesions. The incidence of the abnormalities was similar in all dose groups and both sexes, and therefore does not appear to be treatment-related.Body WeightBody weight differences between the treated and control animals of both sexes were slight during the course of the study. No consistent pattern related to the administration of the test substance was evident. It is concluded by the authors that the dose levels selected did not affect body weights significantly.Food ConsumptionAmong treated males, slight reductions in food consumption were evident during weeks 1-3 as compared to the control. Thereafter, mean food consumption values were comparable in all groups. Mean food consumption values tended to be slightly higher in the treated females as compared to the controls during the course of the study. The increases were slight and are not considered to be of toxicological significance.Test Substance IntakeTest substance intake values based on nominal dietary concentrations were approximately proportional to the dietary concentrations. Test substance intakes were highest for all groups during the early phase of the study and gradually declined reaching a plateau by approximately week 10. Since the females consumed more feed on a body weight basis, their test substance intake calues were highter as compared to the males within a given group.HematologyPretest hematology values were within normal limits for both sexes. Occasional statistically significant differences were noted in the group mean hematology data for the treated animals as compared to the control groups, for both males and females. However, as all means were generally within the normal physiological range and no consistent treatment-related pattern evident, it was concluded by the authors that the administration of the test material did not significantly alter the hematology parameters evaluated.Clinical ChemistryPretest values were normal for both sexes. At month 1, total bilirubin values were significantly decreased in the 2 highest dose groups while total protein and globulin levels were significantly increased in only the highest dose group. Among the females, month 1 alkaline phosphatase was slightly elevated in all but the control and low dose groups. Statistical significance was noted for only the 250ppm group. At Month 3, a trend toward increased SGOT levels was found among the treated males, while BUN levels tended to be lower in dosed animals as compared to the controls. In addition, LDH was significantly increased in the high dose only. Among the females, SGOT, SGPT, and LDH were significantly elevated in the high dose only. In addition, total protein and globulin values were significantly decreased in the high dose. Other significant differences were noted in both sexes at the 1 and 3 month evaluations. However, these differences were generally slight and the values within the normal physiological range for animals of this species and strain. The combination of increases in SGOT, SGPT, and LDH in the males and decreased BUN in the females at Month 3 suggest that the liver may be a target organ. The magnitude of these differences was slight and generally confined to the high dose group. Therefore any alterations in liver function would be expected to be minimal in degree. UrinalysisThere were no effects on urinalysis parameters noted in any animal over the course of the study.Organ Weight and Organ:Body weight ratiosAmong the males, the high dose kidney/body weight ratio was increased, while the absolute liver weights were decreased in the 150 and 250 ppm groups. Among the females, the absolute and relative liver and spleen weights were significantly increased in the 25 and 2500 ppm groups, respectively. The changes were slight and appear due to biological variation as no dose-response pattern emerged. The possible exception is the tendency toward increased liver weights in the treated females. As previously discussed, the clinical biochemistry values suggest the liver as a target organ for the test material. Frequently, liver weight changes are secondary to biochemical altherations in liver morphology and/or function.PathologyGross post-mortem examinations revealed no significant compound-related abnormalities. Lesions noted occurred sporadically and/or were found in both the control and treated animals at approximately the same incidence. Microscopic evaluations revealed patchy hepatocellular vacoulization in the livers of 3/20 males and 4/20 females in the high dose group This finding ranged from nomimal to moderate in degree. These vacuoles, presumably containing lipid, suggest that the liver may be a target organ for the test compound, as the clinical chemistry evaluations showed increased serum concentrations of hepatic enzymes.

Dose descriptor:

LOAEL

Effect level:

2 500 ppm

Sex:

male/female

Basis for effect level:

other: Clinical chemistry parameters (increased blood level of liver enzymes) and pathology of the liver

Dose descriptor:

NOAEL

Effect level:

250 ppm

Sex:

male/female

Basis for effect level:

other: Clinical Chemistry in high dose and Liver pathology in High dose group (2500ppm)

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male

Basis for effect level:

other: Clinical Chemistry in high dose and Liver pathology in High dose group (2500ppm)

Dose descriptor:

NOAEL

Effect level:

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

Sex:

female

Basis for effect level:

other: Clinical Chemistry in high dose and Liver pathology in High dose group (2500ppm)

Critical effects observed:

not specified

Conclusions:

Based on the observations in the high dose group (clinical chemistry parameters and pathology of the liver) it is considered that the High dose group (2500ppm) is the LOAEL for this study. It is clear from the effects observed that AMP targets the liver almost exclusively. Whilst this study was affected by the presence of an eye infection, it presented in all dose groups including control and other than the eye obsevations, it does not appeared to have significantly affected the outcome of the study. However it cannot be ignored that the presence of an eye infection may have stressed the animals leading to an unnatural or more profound response to the administration of AMP.

Reference 4

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline and GLP study but without complete documentation available

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Not available

Route of administration:

oral: unspecified

Vehicle:

not specified

Details on oral exposure:

Not available

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

one per day, 7 days per week

Remarks:

Doses / Concentrations:
4, 20, 100 and 500 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10 or more

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: In a preliminary study to establish the doses for the definitive study, a single dose of 1250 mg.kg bw of the test substance resulted in 40% mortality of female rats. The definitive study high dose was set at 500 mg/kg bw/d

Positive control:

no

Observations and examinations performed and frequency:

no details available

Sacrifice and pathology:

no details available

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

increased salivation in high dose males

Mortality:

mortality observed, treatment-related

Description (incidence):

increased salivation in high dose males

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

acidic urine in high dose males and females

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

NOAEL in the definitive study is 500 mg/kg bw/day.
A preliminary dose-finding study indicated mortality of females at 1250 mg/kg bw

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

54 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Based on one of the components in a worst case approach (the substance consists of 37%w/w oxazolidine and 63%w/w PTSA). Taking into account only the toxicity of 4,4-dimethyl oxazolidine the NOAEL is calculated to be 54 mg/kg bw (based on a NOAEL of 20 mg/kg bw). Taking into account in addition th subactute NOAEL for PTSA of 500 with the standard safety factor for extrapolation to a chronic NOAEL this leads to an overall NOAEL of 60 mg/kg bw (0.37*20 + 0.63*83.3).

System:

hepatobiliary

Organ:

liver

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

Mode of Action Analysis / Human Relevance Framework

The NOAEL from the 2-generation study performed using 4,4-dimethyl oxazolidine is taken as starting point. This substance hydrolyses almost immediately in the stomach following oral dosing; releasing formaldehyde and AMP (refer to summary of reproductive toxicity). In this study the rat the NOAEL was 20 mg/kg bw/day. The primary systemic toxic effect observed was hepatotoxicity at the next highest dose. With respect to extrapolating from the 2-generation study to a long term human exposure situation, a factor of 1 is considered sufficient due to the length of this study, and the consistency of the results with the sub-chronic and chronic rat and dog studies with AMP.

The repeated dose oral toxicity of AMP has been assessed in multiple species (dog and rat), over multiple dosing periods (90 day, 1 year). In both species, oral dosing was well tolerated, although animals dosed in a study where the pH of the test material was 11 showed evidence of significant gastrointestinal irritation and distress due to the high alkalinity of the test material. The liver appears to be the target organ for AMP. Specifically, AMP appears to cause a dose related accumulation of lipids in hepatocytes, and this is linked to an increase in liver weight and presence of liver enzymes in the blood in the more severely affected animals. There appears to be an increased sensitivity to this effect in females compared to males. The dog studies are less adequate to be used in risk assessment, as the dose levels applied were not well chosen and do not allow a conclusion on a NOAEL. However, the results are indicative for a species difference.

In order to account for the species difference seen between dog and rat (and rabbit and rat) in the developmental toxicity studies) an additional safety factor will be included in the derivation of the DNEL based on the rat study with oxazolidine.

Choline and phosphatidyl choline are key in the transport of triglycerides from the hepatocytes in the form of VLDL (very low density lipoprotien). Interfering with the manufacture of phosphatidyl choline thus limits the capacity of the liver to transport lipids. The effect of 4,4-dimethyl oxazolidine (and thus AMP) on the liver appears to be a result of interference with phospholipid synthesis in the hepatocytes. Various publications on AMP from the 1950's and 1960's have identified that it is capable of becoming incorporated into phospholipids in place of ethanolamine and/or choline and that it inhibits the uptake of choline by the liver cells. AMP also appears to inhibit the formation of choline in the liver via the conversion of ethanolamine to choline. It is possible that a phosphatidyl AMP moiety is competing for the enzyme phosphatidyl ethanolamine methyl transferase (converts phosphatidyl ethanolamine to phosphatidyl choline), preventing the formation of phosphatidyl choline, however it is as yet unclear as to the exact mechanism. What is however clear is that in the presence of suficient dietary choline, the effects of AMP on the liver are prevented, and this is likely due to the lower dependance on de novo choline synthesis when sufficient choline is present in the diet. Thus the hepatotoxicity is in part dependent on the presence of sufficient choline in the diet.

 

The transport of lipids from hepatocytes is fairly consistent through out mammalian physiology, i.e. the use of a phospholipid based transport such as VLDL. Therefore the effects observed in rats and dogs are likely relevant to man. However the increase in hepatic lipids is only the first step in the toxicity to the liver, and further indicators of toxicity are only evident after some accumulation of lipids. There is no evidence of liver toxicity (e.g. increased liver weight, increase in liver enzymes in the blood etc.) at doses where lipid accumulation in hepatocytes is not apparent . It is also apparent that the liver toxicity is the most sensitive endpoint, occurring at doses lower than those causing other effects, such as increased post implantation loss (refer to reproductive toxicity section).

Additional information

As described above the contribution of PTSA is expected to be minor compared to the effects of 4,4-dimethyl oxazolidine

Justification for classification or non-classification

No classification for Chronic toxicity or Target Organ toxicity is proposed.

According to the GHS criteria for "specific target organ toxicity", severe fatty change in the liver is considered to be an effect of relevance for classification. However, in all the available studies on AMP and

4,4-dimethyl oxazolidine, the fatty change observed at doses below 100 mg/kg bw in the rat were minor changes and could not be classed as severe. Thus the effect does not meet the criteria for classification.