Alkylbenzenesulphonic acid compound with alkanolamine

Endpoint summary

Description of key information

The acid and amine parts of the compound were tested in repeated dose studies. For the amine an oral 90-day study (Dow 2003) gave a NOAEL of 100 mg/kg bw in males and 500 mg/kg bw in females based on reduced weight of the kidneys. In a dermal 28 day study the NOAEL for systemic toxicity was 1000 mg/kg bw, while slight irritation of the skin was observed at 500 mg/kg bw (NOAEL local effects 100 mg/kg bw) (1993).

For the acid a study according to OECD 422 is available with a NOAEL of 100 mg/kg bw based on local effects on the gastro-intetinal tract. These effects may be related to the acidity of the tested substance. A 90-day feeding test with the sodium salt of the acid gave a NOAEL of 220 mg/kg bw (the highest tested dose) (1965).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

other: the tested substance is the acid of the compound

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well-documented study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

1) Test animals- Supplier: Orient Bio Co. Ltd. 143-1, Sangdaewon-dong, Jungwon-ku, Sungnam, Gyunggi-do, 462-120 Korea- Age at study initiation: 7-week-old animals for male and female- No. of animals at receipt: 57 for male and female- Body weights at study initiation: 212.5 – 243.8 g for males and 147.6 -168.6 g for females- Age at the first day of treatment: 8 weeks for male and female- Body weight range at the first day of treatment: 274.2∼311.1 g for males and 175.7∼213.4 g for females- All animals were visually examined on acquisition. Only the animals remained in good physical condition during the 6-day acclimatization in the animal room were selected for the test.
2) Environmental condition- Temperature 23 +/- 3 deg C, relative humidity of 50 +/- 10%; ventilation of 10 to 20 times/hours; light/dark cycle 12 h/12 h- All animals used in this study were cared for in accordance with the principles outlined in the "Guide for the Care and Use of Laboratory Animals", a NIH publication.
3) Monitoring- Room temperature was generally in the range 20 ~ 26 deg C, relative humidity was generally in the range 40 ~ 60%. No significant deviations, which can affect the experiment, were observed.
4) Housing and identification of animals- Equal or less than five for the quarantine and acclimatization- Equal or less than two for the pre-mating, treatment and recovery period5) Diet, water and bedding material- Pelleted maintenance diet and tap water ad libitum; no contaminants (analysed)

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test article of the highest dose group was mixed with water for injection, and the low dose group's test article was prepared by dilution of that of the highest dose group. The test article solutions were prepared once a day before completion of the analytical method validation, and after completion the test article was formulated over once a week.

Duration of treatment / exposure:

From 2 weeks before mating to the end of the mating period for male (at least 28 days)From 2 weeks before mating to day 4 of lactation including the mating and gestation periods for female- Post exposure period: 15 days in both sexes

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
0, 100, 200, and 400 mg/kg bw/day (Dosing volume 10ml/kg/day)
Basis:
nominal in diet

No. of animals per sex per dose:

10 males and females for 100 and 200 mg/kg bw/day and 16 males and females for 400 mg/kg bw/day (10 was for test group and 6 was for recovery group), 16 males and females for vehicle control (10 was for test group and 6 was for recovery group)

Control animals:

yes

Details on study design:

- Dose levels determined in a pilot toxicity study of dodecylbenzenesulfonic acid in rats- Constant dosage volume of 10 mL/kg bw/day: calculated with Path/Tox system according to the basis of recently measured body weight.- Dosing of both sexes was begun at 2 weeks prior to mating. Dosing was continued in both sexes during the mating period. Males were dosed after the mating period at least until the minimum total dosing period of 28 days had been completed. Daily dosing of the parental females was continued throughout pregnancy and at least up to day 4 post-partum

Statistics:

- Body weights, food consumption, organ weights, and clinical pathology : means the standard deviation of each mean. - Bartlett's test : analyzing for homogeneity of variance- Dunnett's t test : analyzing for the significance of inter-group differences- Analysis of Variance : analyzing for homogeneous data- Kruskal-Wallis test : analyzing for Heterogeneous data- Dunn's Rank Sum test : analyzing for the significance of inter-group differences between the control and treated groups- F test : analyzing the data of recovery groups for homogeneity of variance- Dunnett's t test : analyzing for homogeneous data- Dunn's Rank Sum test : analyzing for the significance of inter-group differences- t test : analyzing for Heterogeneous data- Kruskal-Wallis test : analyzing for the significance of inter-group differences between the control and treated group-Statistical analyses were performed by comparing the different dose groups with the vehicle control group using Path/Tox System. - p<0.05 or p<0.01

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

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

Details on results:

-Clinical signs: Treatment-related clinical signs such as salivation and soft feces were observed in all males of the 400 mg/kg bw/day group during the premating and mating periods. Soiled perineal and liquid feces were observed in 2 males of 400 mg/kg bw/day group during the premating. Salivation and soft feces were observed in 9 and 8 females during the premating and in 8 and 5 during the mating period in the 400 mg/kg bw/day group, respectively. Thin appearance, salivation, staining around mouth, soiled perineal region and soft feces were observed in 1, 10, 1, 1 and 4 females during the gestation period in the 400 mg/kg group, respectively. Thin appearance and salivation were observed in 1 and 10 animals in the 400 mg/kg bw/day group during the lactation period, respectively.

- Body weights and food consumption: In males, a statistically significant decrease was observed on days 8 and 14 of premating. In females, there was no statistically significant changes except a decrease on day 20 of gestation at 400 mg/kg bw/day. In males, a statistically significant decrease in food consumption was observed in the 400 mg/kg bw/day group on days 2 and 9 of the premating, and in females it was observed on day 2 of premating and day 8 of gestation.

- Neurobehavioral evaluation: No treatment-related changes were observed in any of the treatment group.- Urinalysis for males: There were no treatment-related changes for males.

- Hematological test : In hematology test, activated partial thromboplastin time (APTT) was statistically significantly decreased in male of the 400 mg/kg bw/day group. No treatment-related changes were observed in females. In recovery group, a statically significant decrease in RBC count (RBC) was observed in males of the 400 mg/kg bw/day group and an increase in mean corpuscular hemoglobin (MCH) was observed in females.

- Biochemical test: In serum biochemistry test, a statistically significant increase in A/G ratio andalanine aminotransferase (ALT) was observed in males of the 400 mg/kg bw/day group. In females, a statistically significant decrease in blood urine nitrogen (BUN) observed in all treatment groups and an decrease in albumin (ALB) was also observed in the 400 mg/kg bw/day group. In recovery group, a statistically significant decrease in ALT and increase in ALP were observed in females of the 400 mg/kg bw/day group.

- Gross findings: There were no treatment-related changes for all animals

- Organ weights: In males, no a statistically significant changes were observed in any of the treatment groups. In females, a statistically significant increase in absolute weight of ovaries was observed in the 200 mg/kg bw/day group, and a decrease in absolute weight of salivary gland and heart was observed in the 400 mg/kg bw/day groups. The mean weight of ovaries for main group was 0.108 g in compared with vehicle control of 0.093 g and the mean weight of salivary glands for main group was 0.462 g in compared with vehicle control of 0.532 g. Also, the mean weight of heart for main group was 0.816 g in compared with vehicle control of 0.955 g. In recovery groups, a significant decrease in liver and kidney was observed in males the 400 mg/kg bw/day group. No significant differences were observed between vehicle control and test group for organ weights in females.

- Histopathological findings: Squamous cell hyperplasia in stomach was observed in males and females at 200 and 400 mg/kg bw/day. Two cases of minimal forestomach erosion/ulcer were also observed in males at 400 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: local effects to the GI tract

Dose descriptor:

LOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: local effects to the GI-tract

Critical effects observed:

not specified

Conclusions:

Oral administration of the test substance to rats resulted in soft feces, and squamous cell hyperplasia of stomach in both sexes at 400mg/kg bw/day, and liquid feces and soled perineal region, a decrease in body weight and food consumption in males at 400 mg/kg bw/day. In hematology examination, squamous cell hyperplasia of stomach was observed in both sexes at 200 mg/kg bw/day and forestomach erosion/ulcer was observed in males at 400mg/kg bw/day. Based on these effects the NOAEL value was 100 mg/kg bw/day for male and female rats and the LOAEL value was 200 mg/kg bw/day for male and female rats. From these results, the target organ for oral dosing of dodecylbenzenesulfonic acid was considered to be the stomach.

Executive summary:

Repeated oral dosing of the test substance resulted in soft feces, and squamous cell hyperplasia of stomach in both sexes, and liquid feces and soled perineal region, a decrease in body weight and food consumption, forestomach erosion/ulcer in males at 400 mg/kgbw/day, and squamous cell hyperplasia of stomach in both sexes at 200 mg/kgbw/day.Also,activated partial thromboplastin time (APTT) was statistically significantly decreased in male of the 400 mg/kg bw/day group. Inserum biochemistry test, a statistically significant increase in A/G ratio and alanine aminotransferase (ALT) was observed in males of the 400 mg/kg bw/day group.As a result of organ weight test, no a statistically significant changes were observed in any of the treatment groups in males. A statistically significant increase in absolute weight of ovaries was observed in the 200 mg/kg bw/day group, and a decrease in absolute weight of salivary gland and heart was observed in the 400 mg/kg bw/day groups.

Based on results, all findings were completely or partially reversed during the15daysrecovery period. The target organ for oral dosing of the test substance was considered as stomach. The NOAEL and the LOAEL for repeated toxicity of the test substance was considered to be 100 mg/kg bw/day and 200 mg/kg bw/day in both sexes, respectively.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

other: the tested substance is the acid of the compound

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Qualifier:

no guideline followed

Principles of method if other than guideline:

Method: Groups of 10 male and 10 female weanling rats were fed levels of 0.02, 0.1 or 0.5% LAS in a commercial chow for 90 days. Body weights, food consumption, mortality, and several blood parameters were measured periodically during the study and at termination. Autopsy and microscopic examination of the organs was performed at test termination. Organ weights and gross pathological findings were recorded for the liver, kidneys, spleen, gonads, heart, and brain.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: feed

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

90 days

Frequency of treatment:

Ad libitum

Remarks:

Doses / Concentrations:
0.02, 0.1, and 0.5%
Basis:
nominal in diet

No. of animals per sex per dose:

Each group consists of ten males and females, total three groups

Control animals:

yes

Details on study design:

Control group: Ten males and ten females were fed the same diet as test group without test material and calculated weekly.The test and control diets were fed ad libitum.

Sacrifice and pathology:

At the end of the test period all animals were sacrificed by either inhalation, and each was subjected to a complete autopsy.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Details on results:

- Body weight: No statistically significant effects were observed.
- Food consumption: Two males at the 0.02% dietary level died in the early stages of the study. these deaths were attributed to respiratory illness, not to ingestion of the test material.
- Blood studies: No abnormalities were noted on hematologic value.
- Urine analyses: No differences were observed.
- Pathologic value: No gross pathologic findings were observed.
- Organ weight: statistical analyses of organ: body weight and organ : brain weight ratios indicated there were no significant differences related to ingestion of the test substance. data for organ: body ratios are presented in table.

Dose descriptor:

NOAEL

Effect level:

220 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 0.5%

Critical effects observed:

not specified

No adverse effects were found upon the following parameters: growth, food efficiency, survival, haematologic values, and urinary analytical values. In addition, no treatment-related adverse effects were observed in absolute and relative organ weights, nor were gross histopathological changes observed in any of the organs examined.

Conclusions:

The test substance was fed to rats at dietary levels of 0.02%, 0.1%, and 0.5% for 90 days. No adverse effects were found upon the following parameters: growth, food consumption, food utilization, survival, hematologic values, urinary analytical values, organ weights, and organ: body weight ratios. There were no gross or microscopic tissue changes attributable to ingestion of the test material.

Executive summary:

LAS was administered for 90 days in the diet to 10 male/females rats per dose groups. Doses were 0.02%, 0.1% and 0.5% (8.8, 44, 220 mg/kg bw/day). No adverse effects were found upon the following parameters: growth, food efficiency, survival, haematologic values, urinary analytical values, absolute and relative organ weights, gross and histopathological changes.

The NOAEL is 220 mg/kg bw/day, the highest tested dose.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

other: the tested substance is the amine part of the compound

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP/Guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Fischer 344 rats were selected because of their general acceptance, and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.Supplier and LocationCharles River Laboratories Inc. (Raleigh, North Carolina).Age at Study StartApproximately 7 weeks of age.Physical and AcclimationEach animal was evaluated by a laboratory veterinarian to determine their general health status and acceptability for study purposes upon arrival at the laboratory. The animals were housed 2-3 per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for approximately one week prior to the start of the study.HousingAnimals were housed one per cage in stainless steel cages after assignment to the study. The room relative humidity and temperature were maintained within a range of 46.8 - 56.5% and 21 - 24.4 °C, respectively. A 12-hour light/dark photocycle was maintained for the animal room with lights on at 6:00 a.m. and off at 6:00 p.m., and room air was exchanged approximately 12 - 15 times/hour. Cages had wire-mesh floors that were suspended above cageboards and contained a feed crock and a water bottle.Randomization and IdentificationAnimals were stratified by pre-exposure body weight and then randomly assigned to treatment groups using a computer program. Animals placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) which were correlated to unique alphanumeric identification numbers. Feed and WaterAnimals were provided LabDietâ Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Feed and municipal water were provided ad libitum. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provided adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility.

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Dose Levels and JustificationThe high-dose, 1000 mg/kg bw/day, was chosen based on results of a 14-day study drinking water toxicity study of DIPA (McCollister et al., 1981) and is defined in several regulatory guidelines as a limit dose for subchronic toxicity studies. The high-dose was expected to produce minimally decreased water and feed consumption, some body weight depression, and possible renal effects. The mid-and low-dose levels were expected to provide dose response data for any treatment-related effects observed in the high-dose group. The low-dose was expected to be a no-observed-effect level (NOEL).Dose Preparation and AnalysisThe initial preparation of DIPA/drinking water solutions used historical body weight and water consumption data. Subsequently, the concentrations of DIPA in water were adjusted weekly based upon the most recent mean body weight and water consumption data from the combined 90-day dosing and 28-day recovery groups. The drinking water solutions were prepared weekly throughout the study by serially diluting the high-dose with municipal drinking water. The pH of each dose level was adjusted with concentrated HCl to match that of control water. The homogeneity of the low-dose female and the high-dose male drinking water solutions were determined pre-exposure and near the middle and end of the study.StabilityA previous 14-day toxicity study has shown DIPA to be stable for at least 4 days in drinking water at targeted dose levels of 100 (0.097%) and 3000 (2.51%) mg/kg bw/day. Additional stability data was obtained concurrently with the conduct of the present study for the low dose-female (100 mg/kg bw/day) and high-dose male (1000 mg/kg bw/day) solutions under adjusted pH conditions in the presence and absence of rats. Two male and two female rats were provided the test material solutions for 7 days. The solutions were determined to be stable in the presence or absence of rats for 7 days. The solutions provided to rats for 7 days were maintained at room temperature and determined to be stable for at least 25 days.Concentration VerificationAnalyses of all dose levels and the control were conducted pre-exposure, near the mid-point in the dosing period, and at the end of the dosing period. The method used for analyzing the test material in water was high performance liquid chromatography (HPLC) with refractive index detection and external standards.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The 100 mg/kg bw/day female and 1000 mg/kg bw/day male solutions (which had the lowest and highest concentrations used in this study) were determined to be homogeneous, with relative standard deviations for all solutions sampled between 0.177% and 4.70%. DIPA was determined to be stable for 7 days at the 100 mg/kg bw/day female and the 1000 mg/kg bw/day male dose levels, both for samples provided to rats as well as initial solutions. The range of day 7 samples varied from 99.6 to 105% of original concentrations. The samples provided to rats for 7 days were retained at room temperature and determined to be stable for up to 25 days with a range of 94.6 to 107% of original concentrations. The concentrations of DIPA were determined for the control, 100, 500, and 1000 mg/kg bw/day drinking water solutions mixed on 5/7/02, 6/25/02, and 7/30/02 for male and female rats. HPLC analysis with refractive index detection and external standards indicated 96.4 to 108% of the target concentration was obtained for each individual sample. The mean concentrations for each dose level ranged from 99.2 to 104% of targeted concentration. No DIPA was found in the control drinking water.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuous in water

Remarks:

Doses / Concentrations:0, 100, 500, 1000 mg/kg bw/dayBasis:nominal in water

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

Four groups of 10 male and 10 female Fischer 344 rats were given drinking water formulated to supply 0, 100, 500, or 1000 milligrams DIPA per kilogram body weight per day for at least 90 days to evaluate the potential for systemic toxicity (main group). Additional groups of 10 male and 10 female control and high dose groups of rats were given control water for at least an additional 28 days to evaluate the reversibility of potential effects induced during the 90-day dosing period (recovery group). Cage-side clinical observations, detailed clinical observations, eye exams, body weights, feed consumption, water consumption, hematology, clinical chemistry, urinalysis, and organ weights were conducted on all main group animals. In addition, a gross necropsy was conducted with extensive histopathologic examination of tissues. Test material administration for all animals began on May 9, 2002. Main group rats were necropsied on August 7 and 8, 2002, respectively (test days 91 and 92). Recovery group rats were necropsied on September 5, 2002 (test day 120).Male and female F344 rats were received from Charles River Labs, INC. at 7 weeks of age. They were acclimated to the lab for one week prior to dosing. They were housed singly in wire mesh cages, in rooms designed to maintain adequate environmental conditions for rats. Feed and water were provided ad libitum. They were stratified by body weight using a computer program, randomized into dose groups, and identified by a subcutaneously-implanted transponder correlated to unique alphanumeric identification numbers.

Positive control:

no positive control

Observations and examinations performed and frequency:

Twice each day a cage-side examination was conducted and to the extent possible the following parameters were evaluated: skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), animal behavior, moribundity, mortality, and the availability of feed and water. Detailed clinical observations (DCO) were conducted at pre-exposure and weekly throughout the study. The DCO was conducted on all animals, at approximately the same time each examination day according to an established format. The examination included cage-side, hand-held and open-field observations.

Sacrifice and pathology:

Anatomic PathologyFasted rats submitted alive for necropsy were anesthetized by the inhalation of CO2, weighed, and blood samples were obtained from the orbital sinus. Their tracheas were exposed and clamped, and the animals were euthanized by decapitation. A complete necropsy was conducted on all animals by a veterinary pathologist assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary, and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened glass slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened, and the viscera examined. All visceral tissues were dissected from the carcass, re-examined, and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate buffered 10% formalin using a hand-held syringe and blunt needle. The urinary bladder was distended with formalin via transmural injection from a syringe and 21 gauge needle and subsequently ligated at the urethral orifice. The brain, liver, kidneys, heart, adrenal glands, testes, epididymides, uterus, ovaries,thymus, and spleen were trimmed and weighed immediately. The ratios of organ weight to terminal body weight were calculated. Representative samples of tissues as per guidelines were collected and preserved in neutral, phosphate-buffered 10% formalin. Transponders were removed and placed in jars with the tissues.The number of sections from all preserved tissues as per guidelines were processed by standard histologic procedures from control- and high-dose group animals. Paraffin embedded tissues were sectioned approximately 6 μm thick, stained with hematoxylin and eosin and examined by a veterinary pathologist using a light microscope. The following tissues from the remaining groups were processed and histopathologically examined: kidneys, liver, lungs, urinary bladder, and relevant gross lesions.

Other examinations:

All rats were weighed during the pre-exposure period and weekly during the remainder of the study. Body weight gains were also calculated.Feed consumption data were collected at least weekly for all animals.Clinical PathologyBlood samples were collected from the orbital sinus of all fasted animals, anesthetized with CO2, at the scheduled necropsy.HematologySample PreparationBlood samples for a complete blood count from the main group were mixed with ethylenediamine-tetraacetic acid (EDTA). Blood smears were prepared stained with Wright’s stain and archived for potential future evaluation if warranted. Hematologic parameters were assayed using a Technicon H·1E Hematology Analyzer (Bayer Corporation, Tarrytown, New York).AssaysHematocrit (Hct)Hemoglobin (Hgb) concentrationRed blood cell (RBC) countTotal white blood cell (WBC) countPlatelet (PLAT) countDifferential WBC countRBC indices (MCH, MCV and MCHC)CoagulationSample PreparationBlood samples for coagulation from the main group were collected in sodium citrate tubes, centrifuged and plasma collected and assayed using an ACL9000 (Instrumentation Laboratory, Lexington, Massachusetts).AssayProthrombin time (PT)Clinical ChemistrySample PreparationBlood samples from the main group were collected in glass tubes and sera were separated from cells as soon as possible following blood collection. Serum parameters were measured using a Hitachi 914 Clinical Chemistry Analyzer Boehringer-Mannheim, Indianapolis, Indiana).Enzyme Activities of:Alkaline phosphatase (AP)Alanine aminotransferase (ALT)Aspartate aminotransferase (AST)Concentrations of:Albumin (ALB)Cholesterol (CHOL)Creatinine (CREAT)Electrolytes (Na, K, PO4, Cl and Ca)Glucose (GLU)Total bilirubin (TBILI)Total protein (TP)Urea nitrogen (UN)UrinalysisUrine was collected from all non-fasted animals from the main group during the week prior to necropsy by placing each animal overnight in a metabolism cage (~ 16-hour period). Urine was collected in a glass container and the following assays conducted:AssaysColor, appearance and specific gravity (refractometer), and urine volume.Semiquantitative analysis (MultistixÒ Reagent Strips, Bayer Corporation, Elkhardt, Indiana on the Clinitek 200+) of:pHBilirubinGlucoseProteinsKetonesSemiquantitative analysis (MultistixÒ Reagent Strips, Bayer Corporation, Elkhardt,Indiana on the Clinitek 200+) of:pHBilirubinGlucoseProteinsKetonesBloodUrobilinogenUrine was also collected by manual compression of the bladder prior to the necropsy for characterization of the microsediment using a pooled sample from each dose group/sex.

Statistics:

Means and standard deviations were calculated for all continuous data. Body weights, feed consumption, water consumption, organ weights, urine volume, urine specific gravity, clinical chemistry data, coagulation and appropriate hematologic data were evaluated by Bartlett's test (alpha = 0.01; Winer, 1971) for equality of variances. Based on the outcome of Bartlett's test, exploratory data analysis was performed by a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA). If significant at alpha = 0.05, the ANOVA was followed respectively by Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha = 0.05; Hollander and Wolfe, 1973) with a Bonferroni correction (Miller, 1966) for multiple comparisons to the control. The experiment-wise alpha level was reported for these two tests. DCO incidence scores were statistically analyzed by a z-test of proportions comparing each treated group to the control group (alpha = 0.05; Bruning and Kintz, 1987). Data collected at different time points were analyzed separately. Descriptive statistics only (means and standard deviations) were reported for body weightgains, RBC indices, and differential WBC counts. Statistical outliers were identified by a sequential test (alpha = 0.02; Grubbs, 1969), but routinely excluded only from feed and water consumption calculations. Outliers were excluded from other analyses only for documented, scientifically sound reasons. Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) will be greater than the nominal alpha levels. Therefore, the final interpretation of the data considered statistical analyses along with other factors, such as dose-response relationships and whether the results were consistent with other biological and pathological findings and historical control values.

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 specified

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

effects observed, treatment-related

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

There was no mortality noted, no observations made during clinical exams or detailed clinical observations, no notations during ophthalmologic exams, and no statistically-significant changes in body weight or food consumption that were attributed to DIPA toxicity. Water consumption decreased slightly in treated groups, likely due to water aversion than DIPA toxicity. Test material consumption was within 6% of targeted dose levels throughout the study. There was no change in prothrombin times or any hematologic parameters. WBC counts were unaffected. Serum cholesterol, albumin, and phosphorous of males at the high dose were different than controls, but are considered to be of little clinical significance due to the small magnitude of the change. Urine specific gravity was increased for both sexes at 1000 mg/kg bw/day, and urine volume decreased for females at this dose level. Both findings are consistent with decreased water intake, secondary to DIPA administration, and was considered an adaptive effect. The only organ affected by DIPA was the kidney. Absolute and relative kidney weights were both increased in 500 and 1000 mg/kg bw/day dose groups. Absolute kidney weights of males at 100 mg/kg bw/day were increased. There were no gross pathological findings that were attributed to DIPA, nor did histopathological exams reveal any findings. Although there were no effects attributed to DIPA during the 90 days, the kidneys of the recovery group were examined. Only slight renal tubular degeneration with regeneration was found, with the incidence similar between controls and groups given 1000 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: see 'Remark'

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

LOAEL = 1000 mg/kg bw/day The only effect found in the 500 mg/kg bw/day group was increased relative and absolute kidney weights without any histopathologic correlate. Therefore, NOAEL = 500 mg/kg bw/day for femalesNOAEL = 100 mg/kg bw/day for males

Executive summary:

Ten male and ten female Fischer 344 rats per group were given drinking water formulated to supply 0, 100, 500, or 1000 milligrams of the test substance per kilogram body weight per day (mg/kg/day) for at least 90 days to evaluate the potential for systemic toxicity of

the test substance.

Standard toxicologic parameters were evaluated during the in-life phase of the study with clinical and anatomic pathology investigations at termination. Additional groups of ten/sex were maintained on untreated drinking water for at least an additional 28 days after initially receiving the control or high-dose water (0 or 1000 mg/kg bw/day) for at least 90 days to assess recovery from effects induced by DIPA. Rats of either sex given 1000 mg DIPA/kg bw/day for at least 90 days had few effects, all of which were of minimal degree, attributed to

the test substance.

. While there were no treatment-related clinical signs, rats given this dose level drank slightly less water (females had a greater decrement than males) with corresponding decrements of feed consumption and body weights considered secondary to the water aversion. The body weight decrements were only ~ 2% at termination and were never statistically identified. Urine specific gravity was increased in both sexes while urine volume was decreased for females; both of which were adaptive effects to the decreased water consumption. Serum cholesterol was slightly increased while serum phosphorus was slightly decreased for male rats given 1000 mg/kg bw/day. These effects were not present after the 28-day recovery period. Kidney weights, both absolute and relative to body weight, were increased with the males affected to a greater degree (male relative kidney weight increased ~ 21% and female increased ~ 14%). However, histopathologic effects were not found. After four weeks drinking untreated water, the increase in kidney weight was about one-half of that present at the end of the dosing period for both males and females. This limit dose level, 1000 mg/kg bw/day, was considered to be a LOAEL. The only effect found in rats given 500 mg/kg bw/day was increased absolute and relative kidney weights with males again having a greater increase than females (male relative kidney weight increased ~ 12% and female increased ~ 7%), also without any histopathologic correlate. The absolute kidney weight of males given 100 mg/kg bw/day was also increased and was statistically identified; however, these rats weighed more than controls and the relative kidney weight was similar to controls. Thus, the 500 mg/kg bw/day dose level was considered the NOAEL for females, while 100 mg/kg bw/day was the NOAEL for males.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

in a worst case approach the study on the amine is taken as a representative for the compound.

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

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

other: the tested substance is the amine of the compound

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well-documented publication which meets basic scientific principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Principles of method if other than guideline:

For the dermal toxicity study, 0, 100, 500 or 750 mg DIPA/kg/day was applied to an approximately 2x2 cm interscapular-dorsal area of groups of five Fischer 344 rats/sex/dose level 5 days/week for 4 weeks. Application site skin was clipped free of hair, covered with an occlusive wrap during the dosing period each day, and was reclipped as necessary during the dosing period. The wraps were removed after 6 h and the treated sites were washed by gentle padding with watersoaked gauze. Skin site of application was graded at the end of each week and the dosing period for erythema and eschar, edema, scaling and fissuring, and presence of scabs using a modification of the scoring system recommended by OECD (1981).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344/DuCrj

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River Laboratories Inc. (Kingston, New York and Raleigh, North Carolina)- Diet: LabDiet #5002 Certified Rodent Diet (PMI Nutrition International, St. Louis, MO) ad libitum- Water: tap water ad libitumENVIRONMENTAL CONDITIONS- Temperature (°C): 21-25 °C- Humidity (%): 45-60 %- Air changes (per hr): 12-15- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

TEST SITE- Area of exposure: 2x2 cm- Time intervals for shavings or clipplings: at the beginning and also reclipped as necessary during the dosing period.REMOVAL OF TEST SUBSTANCE- Washing (if done): by gentle padding with watersoaked gauze.- Time after start of exposure: 6 h

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

5 days/week

Remarks:

Doses / Concentrations:0, 100, 500, 750 mg/kg bw/dayBasis:nominal per unit body weight

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes - Time schedule: twice dailyDETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: weeklyBODY WEIGHT: Yes- Time schedule for examinations: weeklyFOOD CONSUMPTION: - Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes WATER CONSUMPTION: Yes- Time schedule for examinations: weeklyHAEMATOLOGY: Yes - Time schedule for collection of blood: at necropsy- Anaesthetic used for blood collection: Yes with methoxyflurane or CO2- Animals fasted: No data- How many animals: allCLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: at necropsy- Animals fasted: No data- How many animals: allStandard hematologic and clinical chemistry parameters were evaluated consistent with globally accepted regulatory guidelines (EEC, 1992; EPA, 1998; OECD, 1998; MITI, 1988).URINALYSIS: Yes - Time schedule for collection of urine: near end of study- Metabolism cages used for collection of urine: No data- Animals fasted: NoStandard urinalysis parameters were evaluated (EEC, 1992; EPA, 1998; OECD, 1998; MITI, 1988).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes HISTOPATHOLOGY: Yes; Histopathologic examination was conducted on selected organs

Statistics:

Consistent with the variety of data collected, a large number of statistical methods were employed. Means and standard deviations were calculated for all continuous data. These parameters were first examined for equality of variance using Bartlett’s test (a = 0.01; Winer, 1971). If the results of the Bartlett’s test were significant, the data were transformed in an attempt to obtain equality of variances. The order of transformations used was the common log, the inverse and the square root with the best fit used for subsequent statistical testing. Weekly body weights were analyzed using a 3-way repeated ANOVA, with body weights the repeated measure and Dunnett's test for comparison to controls; terminal body weight organ weights, clinical pathology parameters and urine specific gravity were evaluated using 2-way ANOVA, separate 1-way ANOVA for each sex if sex by dose significant Dunnett's test for comparison to controls. As statistical interactions (i.e., time by dose or sex by dose) were identified in several of the tests, the alpha levels for interaction terms were set a priori at 0.01–0.10, with Bonferroni’s correction. The alpha level for comparison of individual dose groups to controls was set a priori at 0.05.

Details on results:

CLINICAL SIGNS AND MORTALITYAll rats survived the study period and there were no treatment-related effects.BODY WEIGHT AND WEIGHT GAINno treatment related effects.FOOD CONSUMPTIONno treatment related effects.WATER CONSUMPTIONno treatment related effects.HAEMATOLOGYno treatment related effects.CLINICAL CHEMISTRYno treatment related effects.URINALYSISno treatment related effects.ORGAN WEIGHTSno treatment related effectsGROSS PATHOLOGYerythema was noted for all five males and three females treated with 750 mg/kg/day and two males and two females treated with 500 mg/kg/day. The erythema was graded as very slight (barely perceptible) for all rats except for one female treated with 500 mg/kg/day and one treated with 750 mg/kg/day in which erythema was graded as slight (well-defined) at one time during the study.HISTOPATHOLOGY: NON-NEOPLASTICslight hyperkeratosis of the treated site in all rats given 750 mg/kg/day and very slight hyperkeratosis in two males and two females given 500 mg/kg/day.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

750 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: no systemic effects were reported

Dose descriptor:

NOAEL

Remarks:

local toxicity

Effect level:

100 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: dermal irritancy (100 mg/kg bw/day corresponds to 0.8 mg/cm2 assuming a body weight of 0.2 kg and as 25 cm2 skin was exposed)

Critical effects observed:

not specified

Effects of DIPA applied dermally to F344 rats for 4 weeks:

Parameter	Dose level (mg/kg/day)
	males				females
	0	100	500	750	0	100	500	750
Terminal body weight (g)	213.7	219.7	218.7	213.2	135.2	137.9	135.6	136.1
Serum urea nitrogen (mg/dL)	20	19	20	20	17	17	16	18
Kidney weight (g)	1.661	1.724	1.703	1.676	1.116	1.113	1.119	1.127
rel. kidney weight (g/100g)	0.777	0.786	0.779	0.786	0.826	0.808	0.826	0.828
Histopathology skin - treated site(no examined)	5	5	5	5	5	5	5	5
Within normal limit	5	5	3	0	5	5	3	0
Hyperkeratosis	0	0	0	5	0	0	0	5
Very slight	0	0	2	0	0	0	2	0
Slight	0	0	0	5	0	0	0	5

No statistically significant differences identified (histopathology not statistically analyzed).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The amine did not show systemic effects after dermal application. In view of the toxicokinetics its is expected that if uptake would take place the amine may be the candidate with the highest chance of uptake (see toxicokinetic assessment)

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

other: the tested substance is the amine of the compound

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well-documented publication which meets basic scientific principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Principles of method if other than guideline:

For the dermal toxicity study, 0, 100, 500 or 750 mg DIPA/kg/day was applied to an approximately 2x2 cm interscapular-dorsal area of groups of five Fischer 344 rats/sex/dose level 5 days/week for 4 weeks. Application site skin was clipped free of hair, covered with an occlusive wrap during the dosing period each day, and was reclipped as necessary during the dosing period. The wraps were removed after 6 h and the treated sites were washed by gentle padding with watersoaked gauze. Skin site of application was graded at the end of each week and the dosing period for erythema and eschar, edema, scaling and fissuring, and presence of scabs using a modification of the scoring system recommended by OECD (1981).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344/DuCrj

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River Laboratories Inc. (Kingston, New York and Raleigh, North Carolina)- Diet: LabDiet #5002 Certified Rodent Diet (PMI Nutrition International, St. Louis, MO) ad libitum- Water: tap water ad libitumENVIRONMENTAL CONDITIONS- Temperature (°C): 21-25 °C- Humidity (%): 45-60 %- Air changes (per hr): 12-15- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

TEST SITE- Area of exposure: 2x2 cm- Time intervals for shavings or clipplings: at the beginning and also reclipped as necessary during the dosing period.REMOVAL OF TEST SUBSTANCE- Washing (if done): by gentle padding with watersoaked gauze.- Time after start of exposure: 6 h

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

5 days/week

Remarks:

Doses / Concentrations:0, 100, 500, 750 mg/kg bw/dayBasis:nominal per unit body weight

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes - Time schedule: twice dailyDETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: weeklyBODY WEIGHT: Yes- Time schedule for examinations: weeklyFOOD CONSUMPTION: - Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes WATER CONSUMPTION: Yes- Time schedule for examinations: weeklyHAEMATOLOGY: Yes - Time schedule for collection of blood: at necropsy- Anaesthetic used for blood collection: Yes with methoxyflurane or CO2- Animals fasted: No data- How many animals: allCLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: at necropsy- Animals fasted: No data- How many animals: allStandard hematologic and clinical chemistry parameters were evaluated consistent with globally accepted regulatory guidelines (EEC, 1992; EPA, 1998; OECD, 1998; MITI, 1988).URINALYSIS: Yes - Time schedule for collection of urine: near end of study- Metabolism cages used for collection of urine: No data- Animals fasted: NoStandard urinalysis parameters were evaluated (EEC, 1992; EPA, 1998; OECD, 1998; MITI, 1988).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes HISTOPATHOLOGY: Yes; Histopathologic examination was conducted on selected organs

Statistics:

Consistent with the variety of data collected, a large number of statistical methods were employed. Means and standard deviations were calculated for all continuous data. These parameters were first examined for equality of variance using Bartlett’s test (a = 0.01; Winer, 1971). If the results of the Bartlett’s test were significant, the data were transformed in an attempt to obtain equality of variances. The order of transformations used was the common log, the inverse and the square root with the best fit used for subsequent statistical testing. Weekly body weights were analyzed using a 3-way repeated ANOVA, with body weights the repeated measure and Dunnett's test for comparison to controls; terminal body weight organ weights, clinical pathology parameters and urine specific gravity were evaluated using 2-way ANOVA, separate 1-way ANOVA for each sex if sex by dose significant Dunnett's test for comparison to controls. As statistical interactions (i.e., time by dose or sex by dose) were identified in several of the tests, the alpha levels for interaction terms were set a priori at 0.01–0.10, with Bonferroni’s correction. The alpha level for comparison of individual dose groups to controls was set a priori at 0.05.

Details on results:

CLINICAL SIGNS AND MORTALITYAll rats survived the study period and there were no treatment-related effects.BODY WEIGHT AND WEIGHT GAINno treatment related effects.FOOD CONSUMPTIONno treatment related effects.WATER CONSUMPTIONno treatment related effects.HAEMATOLOGYno treatment related effects.CLINICAL CHEMISTRYno treatment related effects.URINALYSISno treatment related effects.ORGAN WEIGHTSno treatment related effectsGROSS PATHOLOGYerythema was noted for all five males and three females treated with 750 mg/kg/day and two males and two females treated with 500 mg/kg/day. The erythema was graded as very slight (barely perceptible) for all rats except for one female treated with 500 mg/kg/day and one treated with 750 mg/kg/day in which erythema was graded as slight (well-defined) at one time during the study.HISTOPATHOLOGY: NON-NEOPLASTICslight hyperkeratosis of the treated site in all rats given 750 mg/kg/day and very slight hyperkeratosis in two males and two females given 500 mg/kg/day.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

750 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: no systemic effects were reported

Dose descriptor:

NOAEL

Remarks:

local toxicity

Effect level:

100 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: dermal irritancy (100 mg/kg bw/day corresponds to 0.8 mg/cm2 assuming a body weight of 0.2 kg and as 25 cm2 skin was exposed)

Critical effects observed:

not specified

Effects of DIPA applied dermally to F344 rats for 4 weeks:

Parameter	Dose level (mg/kg/day)
	males				females
	0	100	500	750	0	100	500	750
Terminal body weight (g)	213.7	219.7	218.7	213.2	135.2	137.9	135.6	136.1
Serum urea nitrogen (mg/dL)	20	19	20	20	17	17	16	18
Kidney weight (g)	1.661	1.724	1.703	1.676	1.116	1.113	1.119	1.127
rel. kidney weight (g/100g)	0.777	0.786	0.779	0.786	0.826	0.808	0.826	0.828
Histopathology skin - treated site(no examined)	5	5	5	5	5	5	5	5
Within normal limit	5	5	3	0	5	5	3	0
Hyperkeratosis	0	0	0	5	0	0	0	5
Very slight	0	0	2	0	0	0	2	0
Slight	0	0	0	5	0	0	0	5

No statistically significant differences identified (histopathology not statistically analyzed).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.8 mg/cm²

Study duration:

subacute

Species:

rat

Quality of whole database:

ONly a study on the cationic part of the salt was available

Additional information

In a worst case approach the oral NOAEL of 100 mg/kg bw is chosen as starting point for the risk assessment for systemic effects. The order of magnitude of the NOAELs for the acid and the amine is the same. There is no reason to believe that a combined effect due to exposure of the cation and anion will occur, therefore the lowest NOAEL is chosen.

Based on the outcome of the 28 -day dermal study and taking into account that the acid may have irritant properties due to its acidity, the NOAEL for local effects found for the amine will be the starting point (NOAEL 0.8 mg/cm3).

Justification for classification or non-classification

Based on the available information the salt does not need to be classified according to CLP (Regulation EC No 1272/2008)