Registration Dossier

Administrative data

Description of key information

- Subacute (28 day) study; oral (gavage), rat (Charles River CD; 25/sex/dose), OECD guideline 407, GLP; NOAEL = 500 mg/kg bw/d (highest dose level tested); read across from MDEA-Esterquat C16-18 and C18 unsatd.

- Subchronic (91 day) study; oral (gavage), rat (Crl CD BR; 15/sex/dose), OECD guideline 408, GLP; NOAEL = 500 mg/kg bw/d (highest dose level tested); read across from MDEA-Esterquat C16-18 and C18 unsatd.

- Subacute (28 day) study; oral (gavage), rat (Crl:WI(Han); 3/sex/dose), similar to OECD guideline 407, GLP, bridging study; NOAEL 1000 mg/kg bw/day; read-across from MDIPA Esterquat C18 unsatd.

- Subchronic (91 day) study; oral (drinking water), rat (Fischer 344/DuCrl; 10/sex/dose), equivalent to OECD guideline 408, GLP; NOAEL = 500 mg/kg bw/d (as DIPA), corresponding to 2850 mg/kg bw/d (as MDIPA-Esterquat C16-18 and C18 unsatd.); read-across from Diisopropanolamine (DIPA)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1992-10-30 to 1993-02-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl CD BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: 4 weeks
- Weight at study initiation: 180-210 g (males) and 162-191 g (females)
- Fasting period before study: None
- Housing: individually in wire-mesh cages (for the first two days of the conditioning period, animals were housed 3/cage to allow for acclimation to the automatic watering system).
- Diet (e.g. ad libitum): ground Certified Rodent Chow #5002, Purina Mills, Inc., St. Louis, MO ad libitum
- Water (e.g. ad libitum): ad libitum, water was analyzed on a quarterly basis for the presence of heavy metals, pesticides and other contaminants
- Acclimation period: 15 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 73.2 +/- 1.0
- Humidity (%): 47.9 +/- 5.8
- Air changes (per hr): Not available
- Photoperiod (hrs dark / hrs light): 12 hours dark/light


IN-LIFE DATES: From: 1992-10-21 To: 1993-02-04
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was prepared in oral dosing suspensions at concentrations of 0.01, 0.1 and 5.0 % to provide dosage levels of 1, 10, and 500 mg/kg/bw/day.

Dosing suspensions of the test substance required modification of the vehicle in order to produce a consistent pH of 2.5. According to the Sponsor, the stock test preparations were pH 2.15. Since the stock suspensions were to be diluted with the pH Control (pH 2.5) to produce the 5 % dosing suspension, minor adjustment of the pH Control (to slightly increase pH with small amounts of 1 N sodium hydroxide) were necessary to insure a final pH of 2.5 for the dosing suspensions. Pre-test experiments were performed with the stock test article suspensions and the pH Control to determine the necessary adjustment.

Following adjustment of the pH Control for use as a diluent, this modified pH Control was mixed with the stock test substance suspensions to produce a 5 % dosing suspension of test substance (high dose) with a pH of approx. 2.5. The exact proportions of stock test substance suspension to modified pH Control varied with variation of test substance in the stock test substance suspension supplied by the Sponsor. The mid-and low-dose suspensions (0.1 and 0.01 %) were then produced via serial dilution of the 5 % dosing suspension with unmodified pH Control. Each phase of the blending and serial dilutions utilized double-rinsed polypropylene equipment. Each suspension was mixed with a magnetic stirring device for a minimum of 15 minutes between steps. The pH of the suspensions was monitored throughout the mixing period.

Final dilutions permitted once daily administration at a rate of 10 ml/kg. Fresh suspensions were prepared as needed (approx. bi-weekly) and stored at ambient temperature. Test substance suspensions were agitated with magnetic mixers for a minimum of 10 minutes each day immediately prior to dosing.


DIET PREPARATION
- Rate of preparation of diet (frequency): Not available
- Mixing appropriate amounts with (Type of food): Not available
- Storage temperature of food: Not available


VEHICLE: deionized water
- Justification for use and choice of vehicle (if other than water): Not available
- Concentration in vehicle: Not available
- Amount of vehicle (if gavage): Not available
- Lot/batch no. (if required): Not available
- Purity: Not available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test substance were collected on study days 25, 46, 77, and 92 for stability analysis; and on study days 25, 49, and 77 for concentration analysis. Duplicate samples were taken on days 25, 46, 63, 77, and 92 for microbiological characterization. All samples were delivered for analysis to the Sponsor. Stability and concentration samples were sent frozen and microbiological characterization samples were sent at ambient temperatures. All sampling was conducted immediately after active agitation of the suspensions.

pH TESTING: Dosing suspenstions and the pH Control solution were tested for conformance with pH requirements (pH 2.5 +/- 0.3) each day prior to dosing.

STABILITY ANALYSIS: These samples were collected prior to changeover to fresh test substance and dosing suspensions. They represent material and preparations in use for the preceding, approximate bi-weekly period. Samples were shipped frozen.

CONCENTRATION ANALYSIS: Samples of all dosing preparations and controls were taken on study days 1, 26, 49 and 77 for concentration analysis by the Sponsor. These samples were collected from freshly produced dosing suspensions prepared from recently received test material. Samples were shipped frozen.

MICROBIOLOGICAL CHARACTERIZATION: Samples of the dosing preparations and controls were taken on study days 25, 46, 63, 77, and 92 for microbiological characterization by the Sponsor. Similar to stability samples, those taken for microbiological characterization represented test substance and preparations in use for the proceeding, approximate bi-weekly period. These samples were shipped at ambient temperatures.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily, 7 days/week, for 13 weeks
Remarks:
Doses / Concentrations:
500 mg/kg/bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
10 mg/kg/bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1 mg/kg/bw/day
Basis:
actual ingested
No. of animals per sex per dose:
15 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Oral gavage was used to ensure delivery of a controlled and consistent dose of the test substance. Dosage levels were selected based on data generated from a 14-day (range-finding) toxicity study in rats (IRDC 191-1542), and the Sponsor's experience with subchronic
studies for structurally similar compounds. The volume administered to each rat (10 ml/kg) was adjusted based on the most recent body weight.
- Rationale for animal assignment (if not random): Prior to randomization into study groups, the animals were weighed, sexed and examined for evidence of disease and other physical abnormalities. After eliminating animals based on these criteria, animals found to be acceptable for study use
were randomized utilizing a block randomization procedure in which animals were stratified by body weight. Homogeneity of group variance by
body weight was used as the criterion for acceptance, at which time the randomization was accepted and permanent animal numbers were assigned.
- Rationale for selecting satellite groups: NA
- Post-exposure recovery period in satellite groups: NA
- Section schedule rationale (if not random): NA
Positive control:
No data
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (mortality)
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Rats were observed twice daily for signs of overt toxicity at the times of the mortality/morbidity checks. Detailed clinical examinations were also performed at least weekly and included evaluations of appearance and condition, behaviour and activity, excretory function, respiration, orifices, eyes and palpable masses.


BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were obtained during the pretest period and weekly during the study and prior to termination.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
-Individual food consumption was calculated weekly during the study.


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:N/A


OPHTHALMOSCOPIC EXAMINATION: Yes (performed by a veterinary ophthalmologist using a Keeler Indirect Ophthalmoscope of the cornea, sclera, iris and fundus)
- Time schedule for examinations: during acclimation period and prior to terminal sacrifice
- Dose groups that were examined: all animals


HAEMATOLOGY: Yes
- Time schedule for collection of blood: taken at study termination
- Anaesthetic used for blood collection: Yes (CO2). Samples obtained via cardiac puncture
- Animals fasted: Yes, overnight. Animals had free access to water prior to blood collection. Urine was collected during the fasting period.
- How many animals: 10 animals/sex/group at termination. Studies from 6 animals scheduled for coagulation testing clotted prior to analysis. Blood samples were collected from 6 additional animals for coagulation testing in order to complete the 10/animals/sex/group protocol requirement.
- Parameters examined: leukocyte count, erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelets, differential leukocyte count, prothrombin time (PT), activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: taken at study termination.
- Animals fasted: Yes, overnight. Animals had free access to water prior to blood collection. Urine was collected during the fasting period.
- How many animals: 10 animals/sex/group at termination. Studies from 6 animals scheduled for coagulation testing clotted prior to analysis. Blood samples were collected from 6 additional animals for coagulation testing in order to complete the 10/animals/sex/group protocol requirement.
- Parameters examined: sodium, potassium, chloride, calcium, inorganic phosphorous, alkaline phosphatase, total bilirubin, gamma glutamyl transpeptidase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen, creatinine, total protein, albumin, cholesterol, glucose.


URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected during the fasting period before blood was drawn.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: specific gravity and volume.


NEUROBEHAVIOURAL EXAMINATION: No



OTHER: Not available
Sacrifice and pathology:
All animals received a complete postmortem examination under the direct supervision of a pathologist. All animals were necropsied in a replicate order, and a necropsy body weight was obtained immediately following euthanization, but before exsanguination. This weight was used to determine the organ to body weight ratios. All animals were euthanized by methoxyfluorane anesthesis.

GROSS PATHOLOGY: Yes
After a thorough external examination, the skin was reflected from ventral midline incision and any abnormalities were identified and correlated with antemortem findings. The abdominal, thoracic and cranial cavities were examined for abnormalities and the organs removed, examined and, where required by protocol, placed in phosphate-buffered neutral formalin. All macroscopic abnormalities were recorded on the Pathology Record sheet.

Organ weights were determined for the following tissues for all animals and appropriate weight ratios calculated (absolute and relative to body and brain weights). Paired organs were weighed together. Adrenal, brain with stem, kidney, liver, ovary, testis.

HISTOPATHOLOGY: Yes
Representative samples of protocol designated organs and tissues were processed and embedded in paraffin for the preparation and microscopic examination of stained sections for all animals in the pH Control and 500 mg/kg/bw/day dosage groups.

A four-step grading system of trace, mild, moderate, and severe was used to define gradable lesions for comparison between dosage groups. Representative samples of the following tissues were collected and examined microscopically: adrenal, aorta, bone marrow smear, brain, exoribital lacrimal gland, eye including optic nerve, femur, GI tract, gonads, gross lesions, heart, kidney, liver, lung with bronchi, lymph nodes, mammary gland (females), pancreas, pitituary, prostate and seminal vesicle, salivary gland, sciatic nerve, skeletal muscle (thigh), skin, spinal cord, spleen, sternum (with bone marrow), thymus, thyroid, parathyroid, trachea, urinary bladder, uterus and cervix, vagina.
Other examinations:
Not available
Statistics:
Body weight, food consumption, clinical pathology laboratory, and organ weights (absolute and relative) were analyzed using analysis of variance (one-way classification) and Bartlett's test for homogenecity of variance. Treatment groups were compared to the pH Control group, by sex, using appropriate t-statistic (for equal or unequal variance) as described by Steal and Torrie. Dunnett's multiple comparison tables or pairwise comparisons with a Bonferroni correction were used to determine the significance of differences. Non parametric analyses were conducted as appropriate by transforming the data into ranks prior to analysis as described by Conover and Iman. All statistical analyses were performed with P less than or equal to 0.05 and P less than or equal to 0.01 used as levels of significance.
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):
not specified
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 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:
CLINICAL SIGNS AND MORTALITY: All rats survived to study termination. No test substance-related signs of overt toxicity were observed during the study. Clinical examinations of the animals did not produce any evidence of test substance-related effects.


BODY WEIGHT AND WEIGHT GAIN: No significant differences in body weights or body weight increases occurred over the course of the study.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No significant differences in food consumption occurred over the course of the study in any of the groups.


FOOD EFFICIENCY: Not available


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not available

OPHTHALMOSCOPIC EXAMINATION: No test substance-related ophthalmoscopic abnormalities were detected; the observations noted were representative of pathology that would be expected for this group of rats considering age, sex and strain.


HAEMATOLOGY: Mean MCV values were statistically significantly lower (p less than or equal to 0.05) in male rats in the water control, 10 mg/kg/bw/day, and 500 mg/kg bw/day treatment groups as compared to the pH Control group. The mean MCV values in these groups were within +/- 2 standard deviations of this laboratory's historical control mean value for this parameter for rats of this strain, age, and sex. The mean MCV values in these groups also fall within published reference ranges for rats of this strain, age, and sex. Because of these considerations, because this change was not present in both sexes, and because this change also occurred in the water control group, this haematological change, although statistically significant, was not considered to be toxicologically significant or test article-related.


CLINICAL CHEMISTRY: (Blood Chemistry) Mean urea nitrogen values were statistically significantly lower (p less than or equal to 0.01) in male rats in the 500 mg/kg bw/day treatment group as compared to the pH Control group. The mean urea nitrogen value in this group was within +/- 2 standard deviations of the testing laboratory's historical control mean value for this paramater for rats of this strain, age, and sex. Because of the small magnitude of the change, because this change was not present in both sexes, and because no other clinical, biochemical, or pathologic alterations correlating with decreased urea nitrogen were observed, this serum biochemical change, although statistically significant, was not considered to be toxicologically significant or test substance-related.


URINALYSIS: There were no changes that were considered to be toxicologically significant or test substance-related.


NEUROBEHAVIOUR: Not available


ORGAN WEIGHTS: There were no test substance-related changes in the mean absolute weights or mean relative organ weight ratios for the brain, adrenal, kidney, liver and testis of male and female rats.

There was a statistically significant decrease in the mean ovary/body weight ratio for female rats in the 500 mg/kg/bw/day group when compared with the pH Control. This ratio change was, at least in part, due to the variability in the mean body weights with the pH Control being the lowest of all the female groups and the 500 mg/kg bw/day group being the highest of the female groups on the study. There were no statistically significant changes in the mean absolute ovary weight and the ovary/body or the ovary/brain weight ratios when the 1, 10 and 500 mg/kg bw/day treatment groups were compared with the water control or when the same parameters of the ovary from the water control group were compared to the pH Control. The mean absolute ovary weight of the 500 mg/kg bw/day treatment group falls within the range of mean absolute ovarian weight fo 13-week CD rat studies at the testing laboratory. For these reasons, the decreased mean ovary/body weight ratio observed in this study was considered not to be biologically relevant and not test substance-related.


GROSS PATHOLOGY: There were no test substance-related macroscopic findings in either male or female rats necropsied 13 weeks following oral intubation with Water or pH Control material or with 1, 10 or 500 mg/kg bw/day of test article.

No rats died during the duration of the study. A few macroscopic findings were noted at the terminal necropsy. The majority of these macroscopic findings were confirmed microscopically. These findings were considered to be incidental and usual for rats of this age and strain and not test article-related.


HISTOPATHOLOGY: NON-NEOPLASTIC: There were no test substance-related microscopic findings in either male or female rats necropsied following 13 weeks oral intubation.

All macroscopic observations (except cystic ovary, # 41229) correlated with microscopic findings. There were several microscopic findings. These findings were considered to be incidental and usual for rats of this strain and age. The incidence of a given lesion was small and/or occurred with equivalent frequency in treated animals versus controls. There was no evidence of any infectious disease present in any of the rats that would adversely affect the results of the study.

The tissues available for microscopic examination were of satisfactory quality and quantity to adequately evaluate this study. Relevant in-life and necropsy data were available to the study pathologist and were considered in interpretation of the pathology findings. All reference to pathology interpretations in the final report were consistent.


HISTOPATHOLOGY: NEOPLASTIC (if applicable): Not available


HISTORICAL CONTROL DATA (if applicable): Not available


OTHER FINDINGS: Not available
Dose descriptor:
NOAEL
Effect level:
> 500 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: There were no significant adverse test substance-related effects in this study for any of the parameters measured.
Critical effects observed:
no

Statistically significant lower mean MCV values were noted between the 10 mg/kg bw/day, 500 mg/kg bw/day males and the water control male group when compared to the pH control water male group. These differences were not considered biological or test substance related as there were within + or - two standard deviations of the laboratory historical control mean values for rats of this strain, age and sex. In addition, they were within the standard published reference ranges.

The blood urea nitrogen values of the 500 mg/kg bw/day group was significantly lower than the pH control group in male rats. This differences were not considered test substance related as they were within + or - two standard deviatiosn of the laboratory historcal control mean values for rats of this strain, age and sex.

The ovary/body weight ratios of the 500 mg/kg bw/day female rats were signficant lower than the pH control females. The differences in the ovary/body weight ratios were not considered test substance related because of the variability in the mean body weights of pH control group, lowest of all female groups. In addition, there were no test related difference observed in the mean absolute ovary weight of the 500 mg/kg bw/day group in comparison to the other test or control groups and the mean ovary weight also fall with

within + or - two standard deviations of the laboratory historical control mean values for rats of this strain, age and sex.

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected.

Conclusions:
The test substance was administered orally via gavage daily to rats at dosage levels of 1, 10, and 500 mg/kg bw/day for a period of 13 weeks. No biologically significant adverse effects were observed in any test group. The NOAEL was determined to be the high dose level of 500 mg/kg bw/day.
Executive summary:

In a subchronic toxicity study comparable to OECD guideline 408, MDEA-Esterquat C16-18 and C18 unsatd.  (10 % a.i.) was administered to 15 Charles River CD rats / sex/ dose by gavage at dose levels of 1, 10 and 500 mg/kg bw/ day for a period of 13- weeks. One control group received the vehicle, deionized water, and a second control group received pH-adjusted, deionized water (pH 2.5). The regimen for both control groups was identical to treatment groups.

The following parameters were monitored during the study: clinical observations (detailed, weekly; mortality, morbidity, and overt signs of toxicity, twice a day); body weights (weekly); food consumption (weekly); clinical pathology (haematology, blood chemistry, and urinalysis; at termination); opothalmoscopic examinations (once pre-test and prior to sacrifice); macroscopic pathologic examination; absolute and relative organ weights; microscopic pathology.

There were no changes in any of these parameters that were considered to be toxicologically significant or test-substance related.

Reproductive parameters:

Absolute and relative organ weights of ovary and testis and histopathology of reproductive organs (gonads, mammary gland (females only), prostate and seminal vesicle, uterus with cervix and vagina) revealed no test substance related findings.

There is no evidence for specific target organ toxicity in this study.

The no effect level (NOEL) for this study is the high dose level of 500 mg/kg bw/day of the test article.

This subchronic toxicity study in rats is acceptable and satisfies to a large extent the guideline requirement for a subchronic oral study, with exception of highest tested dose. 500 mg/kg bw/day was tested as highest dose instead of 1000 mg/kg/bw/day as recommended by the actual OECD guideline 408.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
OECD guideline studies, or similar to OECD guidelines, no deviations, RL1, GLP

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity data on the target substance MDIPA-Esterquat C16-18 and C18 unsatd. is not available. For the assessment of repeated dose toxicity of MDIPA-Esterquat C16-18 and C18 unsatd. a subacute 28 day oral toxicity study and a subchronic 91 day oral toxicity study conducted with the source substance MDEA-Esterquat C16-18 and C18 unsatd., as well as a 28 day bridging study with the source substance MDIPA Esterquat C18 unsatd. are available. Further support is given by subchronic toxicity data on Diisopropanolamine. A justification for read-across is given below.

 

One 28 day repeated dose study comparable to OECD Guideline 407 and one sub-chronic toxicity study comparable to OECD Guideline 408 are available for the oral route of administration for the source substance MDEA-Esterquat C16-18 and C18 unsatd.

MDEA-Esterquat C16-18 and C18 unsatd. (10 % a. i. aqueous dispersion) was administered by oral gavage to Charles River CD rats, groups of 25 male and female at dose levels of 1, 10 and 500 mg/kg bw/day for 28 days and to15 rats/sex/dose at dose levels of 1, 10 and 500 mg/kg bw/day for a period of 13-weeks. In both studies, limit doses of 1000 mg/kg bw/day as required by the OECD guidelines were not examined. Furthermore, in both studies there were two control groups, animals received either deionised water (water control group) or pH 2.5 adjusted deionised water (pH-water control group).

In both studies all animals survived until study termination and no significant test substance-related adverse findings were detected or observed for any parameter measured.

In the 28 day study three animals exhibited focal scarring or retinal loss. This finding was not considered to be substance-related and no other test article-related ophthalmoscopic abnormalities were detected. All observations noted were pathologies that would be expected for this group of rats considering their age, sex and strain.

A slight but statistically significant decrease in the MCH index was observed in the male high dose group (500 mg/kg bw/day). However, since no corresponding changes were detected in any other haematologic parameter, this change in the MCH was not considered to be test article related.

In the 90 day study a statistically significantly lower mean MCV value was observed in male rates in the water control group and in the 10 and 500 mg/kg bw/day test groups than in the pH-water control group. However, even these lower mean MCV values were within ± 2 standard deviations of laboratory´s historical control mean value for this parameter for rats of this stain, age and sex. Furthermore, they also fall within the ranges of published reference data. Since changes in MCV values were not present in both sexes and since they occurred also in the water control group, they were considered not to be toxicologically significant nor test article-related.

In male rats the blood urea nitrogen values of the 500 mg/kg bw/day group were significantly lower than those of the pH-water control group. These differences were not considered to be test substance-related as there were within the laboratory’s historical control mean values for rats of this strain, age and sex. Individual male and female animals of all groups, including the controls, had elevated values for aspartate aminotransferase (ASAT) activity. These findings in clinical chemistry had no statistical relevance, did not show a dose-response correlation and were without histopathological correlates and therefore they were considered not to be test article-related.

In the 90 day study there were no statistically significant changes in the mean relative ovary/body or ovary/brain weight ratio when compared to both control groups with the exception of a statistically significant decrease in the mean relative ovary/body weight ratio observed in the 500 mg/kg bw/day group when compared to the pH-water control group. The mean body weight of the pH-water control group was the lowest of all female groups in contrast to that of the 500 mg/kg bw/day group, which was the highest. In addition, there were no statistically significant changes in the mean absolute ovary weights. The mean absolute ovary weight of the 500 mg/kg bw/day group falls within the range of the mean absolute ovarian weight for 13-week CD rat studies at the contract laboratory. Therefore, the observed decreased mean ovary/body weight ratio was considered not to be biologically relevant and not test article-related. Individual male and female animals of the 10 mg/kg bw/day group groups, and the control groups, had elevated values for aspartate aminotransferase (ASAT) activity. Also higher alanine aminotransferase (ALAT) activity was seen in individual male and female animals in the 10 mg/kg bw/day group in males and females. These findings in clinical chemistry had no statistical relevance, did not show a dose-response correlation and were without histopathological correlates and therefore they were considered not to be test article-related.

The NOAEL is the highest dose examined, i. e. 500 mg/kg bw/day in both repeated dose toxicity studies. There is no evidence for specific target organ toxicity from both repeated dose toxicity studies.

 

In a subacute toxicity study similar to OECD guideline 407 (2008) and EU method B.7 (2008) MDIPA Esterquat C18 unsatd. (100%) was administered to 3 Crl: WI(Han) rats/sex/dose in propylene glycol by gavage at dose levels of 0, 150, 500, 1000 mg/kg bw/day for 28 consecutive days. As this study was mainly intended for justification of read-across to a structurally comparable substance, the guidelines are not fully applicable. Additional male reproductive parameters have been evaluated.

No toxicologically relevant changes were noted in any of the following parameters investigated in this study: clinical appearance, body weight, food consumption, haematology parameters, macroscopic examination, organ weights, and microscopic examination. The spermatogenic staging profiles were normal in the high dose group males.

Changes were confined to higher alanine aminotransferase activity in two males and one female at 1000 mg/kg and in one male at 500 mg/kg, and higher aspartate aminotransferase activity in one male each at 500 and 1000 mg/kg. These increases did not show a dose-related trend and were not seen among all animals of the same group. Liver weights and macroscopic observations were normal for all animals, including those showing increased alanine/aspartate aminotransferase activity. No further changes in clinical biochemistry parameters (such as increased alkaline phosphatase activity, changes in glucose, total protein/albumin, total bilirubin and cholesterol) were noted that could be indicative of altered liver function. Therefore, the increased ASAT/ ALAT activity is considered of no toxicological relevance and considered indicative of a commonly observed adaptive (hepatic enzyme induction) type of response. Elevated levels of aminotransferases, though neither dose-related nor statistically significant, were also observed in individual animals in the 91-day and 28-day studies with the source substance MDEA-Esterquat C16-18 and C18 unsatd. In those animals with elevated levels of aminotransferases, these findings in clinical chemistry were without histopathological correlates, supporting the presumption of being an adaptive response.

The NOEL in this study based on clinical chemistry was determined to be 500 mg/kg bw/d, the NOAEL was determined to be 1000 mg/kg bw/d.

 

A subchronic toxicity study similar to OECD guideline 408 with Diisopropanolamine (DIPA) is presented as supporting data.

DIPA (98.8 to 99.6%) was administered to 10 Fischer 344/DuCrl rats/sex/dose in drinking water at dose levels of 0, 100, 500, or 1000 mg/kg bw/d daily for 13 weeks. Additional 10 animals in the control and highest dose groups were maintained on untreated drinking water for 4 weeks as recovery group.

No mortality occurred in any group; no treatment related clinical signs were observed. No relevant effects on body weight, ophthalmoscopic examinations, haematological parameters, prothrombin times, gross or histopathology were detected. The water consumption in the high dose group males and females was slightly reduced compared to control animals. Consistent with this, rats of the 1000 mg/kg bw/day dose group had increased urine specific gravity and females had decreased urine volume, which were also considered secondary adaptive effects.

There were minimal, but statistically identified, differences noted for cholesterol (increased), albumin (decreased) and phosphorus (decreased) for males given 1000 mg/kg bw/day. There were no effects upon clinical chemistry parameters for males given 100 or 500 mg/kg bw/day or for females at any dose level. Increased relative kidney weights for males and females of the 500 or 1000 mg/kg bw/day dose group were observed without any treatment-related gross or histopathologic correlate.

In the recovery group, water consumption in the treated group was below control by app. the same degree as at the end of the dosing period; all other affected endpoints showed reversibility. The absolute and relative kidney weights of the treated rats were still above the controls, but the differences were about one-half of those immediately following 13 weeks dosing. No treatment-related renal histopathological changes were observed

The NOAEL in this study was therefore 500 mg/kg bw/d based on minor changes in clinical chemistry in males of the 1000 mg/kg bw/d dose group. The LOAEL was 1000 mg/kg bw/d.

Based on the molecular weights of DIPA (133.19 g/mol) and MDIPA-Esterquat C16-18 and C18 unsatd. (approx. 760 g/mol), the NOAEL of 500 mg/kg bw/d in terms of DIPA has been extrapolated to 2850 mg/kg bw/d in terms of MDIPA-Esterquat C16-18 and C18 unsatd.

 

There are no data gaps for the endpoint repeated dose toxicity. No human data are available. However, there is no reason to believe that these results from rat would not be applicable to humans.

 

Endpoint specific justification for read-across

For details on substance identity, toxicokinetics and detailed toxicological profiles, please refer also to the general justification for read-across given in chapter 5 of the CSR and attached as pdf document to section 7 of the IUCLID file.

 

Structural similarity

a. Structural similarity and functional groups

The target substance, MDIPA-Esterquat C16-18 and C18 unsatd., consists of an amine backbone (MDIPA = Methyldiisopropanolamine) esterified with long chain fatty acids C16, C18 and C18 unsaturated. The main reaction product is the dialkylester compound, next to that small amounts of the monoalkylester may be formed. The amine function is quaternised with two methyl groups. The counter ion is Methosulfate.

The first source substance, MDEA-Esterquat C16-18 and C18 unsatd. , consists of an amine backbone (MDEA = Methyldiethanolamine) esterified with long chain fatty acids C16, C18 and C18 unsaturated. The main reaction product is the dialkylester compound, next to that small amounts of the monoalkylester may be formed. The amine function is quaternised with two methyl groups. The counter ion is Chloride.

The second source substance, MDIPA-Esterquat C18 unsatd., consists of an amine backbone esterified with long chain fatty acids C18 unsaturated. The main reaction product is the dialkylester compound, next to that small amounts of the monoalkylester may be formed. The amine function is quaternised with two methyl groups. The counter ion is Methosulfate.

The source and the target substances share structural similarities with common functional groups (quaternary amines), esters, and fatty acid chains with comparable length and degree of saturation. The amine backbones based on MDEA and MDIPA, respectively, differ only by one methyl group, all functional groups are identical.

 

b. Common breakdown products

The metabolism expected to occur is hydrolysis of the ester-bond by esterases. However, the rate of hydrolysis is assumed to be low. The fraction of metabolised molecules would result in free fatty acids and Dimethyl-DEA (DEA = Diethanolamine) and Dimethyl-DIPA (DIPA = Diisopropanolamine), respectively. The carboxylic acids are further degraded by the mitochondrial beta-oxidation process (for details see common text books on biochemistry). The fatty acids enter normal metabolic pathways and are therefore indistinguishable from fatty acids from other sources including diet. The quaternary ammonium ions are not expected to be further metabolised, but excreted unchanged via the urine.

 

c. Differences

The differences in fatty acid chain length (higher percentage of C16 in the source substance MDEA-Esterquat C16-18 and C18 unsatd.) and degree of saturation (higher degree of unsaturation in the source substance MDIPA-Esterquat C18 unsatd.) may be relevant for local effects (e.g. irritation) but are not considered to be of relevance for repeated dose toxicity.

Chloride is an essential nutrient and present in all organisms; excess chloride is renally excreted (see common textbooks on biology / biochemistry). Methyl sulphate is metabolised to Sulphate and Carbon dioxide, and these are excreted via the urine and released by the lungs, respectively. The anions Chloride and Methyl sulphate are not expected to have any influence on toxicity or reactivity.

The methyl side chain of Dimethyl-DIPA which is not present in Dimethyl-DEA is not expected to enhance reactivity, which is supported by a similar toxicological profile for the source and target substance as well as toxicokinetic data for DEA (Diethanolamine), MDEA, DIPA (Diisopropanolamine) and MDIPA (for details see general justification for read-across.

 

 

Description of interpolation based on structures (see attachment)

 

Source substance 1

Target substance

Source substance 2

MDEA-Esterquat

MDIPA-Esterquat C16-18 and C18 unsatd.

 

MDIPA-Esterquat C18 unsatd.

 

 

OECD guideline 407, rat, oral; NO(A)EL 500 mg/kg bw/day (highest tested dose)
OECD guideline 408, rat, oral; NO(A)EL 500 mg/kg bw/day (highest tested dose)

No data, read-across

OECD guideline 407, rat, oral
bridging study with 3 animals/sex/dose
NOEL 500 mg/kg bw/day; NOAEL 1000 mg/kg bw/day

 

 

Fatty Acid Chain length distribution

<C16 <7%

C16, 16‘, 17 26-35%

C18 42-52%

C18‘ 15-20%

C18‘‘, 18‘‘‘ </= 1.5%

>C18 </= 2%

< C16: <= 6%

C16 - < C18: 20-65%

C18: 4-60%

C18 unsatd.: 10-43%

> C18: <= 5%

<C16 -

C16 </=7%

C18 </= 4%

C18‘ 55-65%

C18‘‘ 18-25%

C18‘‘‘ 6-12%

  >C18 </= 5%

Amine

MDEA

MDIPA

MDIPA

Anion

Chloride

Methyl sulphate

Methyl sulphate

 

 

 

 

 

 

The interpolation of the available data on repeated dose toxicity to fulfil the information requirement for the target substance is based on:

- identical fatty acid chain in the source substance MDEA-Esterquat C16-18 and C18 unsatd. and the target substance MDIPA-Esterquat C16-18 and C18 unsatd.

- identical amine headgroup and counter ion in the source substance MDIPA-Esterquat C18 unsatd. and the target substance MDIPA-Esterquat C16-18 and C18 unsatd.

 

Low systemic toxicity after repeated exposure was shown for both source substances: The NOEL of MDEA-Esterquat C16-18 and C18 unsatd. was 500 mg/kg bw/d in a 28 d study as well as in a 91 d study (highest dose tested in both studies). There is no evidence for specific target organ toxicity from both repeated dose toxicity studies. A 28 d bridging study with MDIPA Esterquat C18 unsatd. with dose levels of 0, 150, 500 and 1000 mg/kg bw/day resulted in a NOEL of 500 mg/kg bw/d and a NOAEL due to changes in clinical chemistry (elevated levels of aminotransferases).Elevated levels of aminotransferases, though neither dose-related nor statistically significant, were also observed in individual animals in the 91-day and 28-day studies with the source substance MDEA-Esterquat C16-18 and C18 unsatd. In those animals with elevated levels of aminotransferases, these findings in clinical chemistry were without histopathological correlates, which supports the presumption of being an adaptive response.

In the repeated dose toxicity studies with the source substance MDEA-Esterquat C16-18 and C18 unsatd. no adverse test substance-related effects were found for any parameter measured, especially regarding organ weights of ovary and testes and histopathology of gonads, uterus, mammary gland, prostate and seminal vesicle. The 28-d bridging study with MDIPA Esterquat C18 unsatd. also included histopathological examinations of the reproductive organs and staging of spermatogenesis. The spermatogenic staging profiles were normal in the high dose group males. No effects on the reproductive organs were noted up to and including 1000 mg/kg bw/d.

The read-across approach is further substantiated by data on the repeated dose toxicity of DIPA. No data is available for the potential metabolite Dimethyl-DIPA itself. To support the absence of toxicity, data on DIPA ,which is a structural part of the target substance MDIPA-Esterquat C16-18 and C18 unsatd. and the source substance MDIPA Esterquat C18 unsatd., is provided.

 

Quality of the experimental data of the analogues

The source substance MDEA-Esterquat C16-18 and C18 unsatd. has been tested in reliable GLP-compliant OECD TG 407 and OECD TG 408 studies up to 500 mg/kg bw/d.

The source MDIPA Esterquat C18 unsatd. has been tested in a GLP compliant 28 d bridging study similar to OECD TG 407 up to 1000 mg/kg bw/d. As this study was mainly intended for justification of read-across to a structurally comparable substance, only 3 animals/sex/dose have been tested. Additional male reproductive parameters have been evaluated.

DIPA has been tested in a GLP compliant study (well-documented publication) equivalent to OECD guideline 408 up to the limit dose of 1000 mg/kg bw/d.

The available data from the source substances are sufficiently reliable to justify the read-across approach.

 

Classification and labelling (Human health)

The source substance MDEA-Esterquat C16-18 and C18 unsatd. is not classified for any human health hazard, whereas the source substance MDIPA Esterquat C18 unsatd. is classified for local effects (irreversible effects on the eye Category 1, irritating to the skin Category 2). The target substance MDIPA-Esterquat C16-18 and C18 unsatd. is classified for local effects (irritating to the eye Category 2, irritating to the skin Category 2). These differences in skin and eye irritation properties are not relevant for repeated dose toxicity as thesedifferences did not have effects on dermal penetration. Dermal penetration rates were equally low for the source substance MDEA-Esterquat C16-18 and C18 unsatd. (<1.4%) and the target substance MDIPA-Esterquat C16-18 and C18 unsatd. (0.3%).

 

Conclusion

The structural similarities between the source and the target substances as presented above support the read-across hypothesis. Adequate and reliable scientific information indicates that the source and target substances have similar toxicity profiles.

The acute oral toxicity for MDEA-Esterquat C16-18 and C18 unsatd., MDIPA-Esterquat C18 unsatd. and MDIPA-Esterquat C16-18 and C18 unsatd. is equally low for all three of them.Reliable data is available for all three substances (see general justification for read-across).

No systemic toxicity was detected in the subacute and subchronic toxicity studies with the source substance MDEA-Esterquat C16-18 and C18 unsatd. (NOEL 500 mg/kg bw/day highest tested dose). In the subacute study with the second source substance,MDIPA-Esterquat C18 unsatd.also no substance related toxicity was detected (NOAEL 1000 mg/kg bw/day). No systemic toxicity was observed in a subchronic toxicity study with DIPA up to a NOAEL of 500 mg/kg bw/day.

Furthermore, based on the available metabolism data as well as on theoretical considerations, source and target substances are expected to follow a similar metabolic pathways leading to rapid metabolic clearance.

The dose descriptor obtained from the existing sub-chronic repeated dose toxicity study performed on the source substance MDEA-Esterquat C16-18 and C18 unsatd. is considered as an appropriate starting point for deriving a DNEL for the target substance MDIPA-Esterquat C16-18 and C18 unsatd. The remaining uncertainty associated with this read-across approach is accounted for by using the appropriate assessment factors, as recommended in ECHA Guidance R.8.

Justification for classification or non-classification

Based on the available data, MDIPA Esterquat C16 -16 and C18 unsatd. does not need to be classified for repeated dose toxicity according to the criteria given in regulation (EC) 1272/2008 or the former European directive on classification and labelling 67/548/EEC. Thus, no labelling is required.