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Description of key information

Key data for subacute toxicity were available from an oral (gavage) OECD 422 study in rats with read-across substance EC 939 -637 -2 or 'Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts' at dose levels given by oral gavage of 100, 300 and 1000 mg/kg bw/day. No relevant effects were observed at 100 and 300 mg/kg bw. At the dose of 1000 mg/kg bw, decreased body weight, increased serum ALAT and decreased serum albumin, macroscopic and microscopic stomach changes and also some lung congestion were observed. NOAEL for systemic toxicity was 300 mg/kg bw/day.

A key 90-day repeated dose toxicity study was also conducted with read-across substance EC 939 -637 -2 or 'Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts'at dose levels of 100, 300, or 1000 mg/kg bw given to rats by daily oral administration via gavage for 90 days, followed by a 4-week recovery period. The NOAEL was 300 mg act.ingr./kg bw/day. Test-item related effects were observed at 1000 mg/kg bw, including a slight reduction in mean body weight in male and female animals, increased drinking water consumption in male and the female animals and changes in the forestomach (non-glandular part of the stomach) of almost all in the form of squamous cell hyperplasia, hyperkeratosis and sometimes submucosal mixed inflammatory cell infiltrate. The body weight and drinking water intake of all previously high-dosed animals was in the normal range during the recovery period, and no abnormalities were noted in the stomach after this recovery period. The changes in the stomach were not considered relevant for humans as humans do not have a forestomach, exposure conditions were considered to be extreme (irritating concentration daily given by bolus) and the findings were completely reversible.

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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
For details please refer to Read Across Justification Document, Section 13.2

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
For details please refer to Read Across Justification Document, Section 13.2

3. ANALOGUE APPROACH JUSTIFICATION
For details please refer to Read Across Justification Document, Section 13.2

4. DATA MATRIX
For details please refer to Read Across Justification Document, Section 13.2
Reason / purpose for cross-reference:
read-across source
Clinical signs:
no effects observed
Description (incidence and severity):
Treatment period: None of the male and female rats treated orally with 100, 300, or 1000 mg act. ingr./kg bw/day revealed any test item-related changes in behaviour, external appearance, consistency of faeces, body posture, and movement and coordination capabilities.
Recovery period: None of the animals previously treated with 1000 mg act. ingr./kg bw/day revealed any abnormalities related to the previous treatment during the 4-week recovery period.
Mortality:
no mortality observed
Description (incidence):
Treatment period No deaths were noted at any dose level. All animals survived until their scheduled sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment period: The mean body weight of the male and female animals treated with 1000 mg act. ingr./kg bw/day was slightly reduced by up to 7.4% in males and up to 10.7% in females in comparison to the control group. The body weight gain and the body weight at autopsy of both sexes were reduced accordingly. This effect is considered to be test item-related.
Recovery period: The body weight of the male and female animals recovered towards the normal range, no noteworthy difference compared to the control group was noted at the end of the recovery period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related toxicologically relevant influence was noted on absolute and relative food consumption.For the male high-dosed animals, a slight but statistically significant (at p ≤ 0.05 or p ≤ 0.01) increase by up to 14.4% was noted for the relative food consumption compared to the control animals starting in test week 2 during the treatment period and lasting until test week 16 during the recovery period, however no significant changes were seen in the absolute food consumption.These changes are within the normal range of biological variation. In test week 17 (end of recovery), when the previously high-dosed male animals revealed no evident difference in the body weight compared to the control animals, there was also no noteworthy difference in the food consumption between the two groups.
For the female animals treated with 1000 mg act. ingr./kg bw/day, a slight but statistically significant (at p ≤ 0.01) decrease in absolute food consumption of up to 14.6% was noted in test weeks 1 and 10 to 12. In test week 1, the relative food consumption was also slightly decreased (statistically significant at p ≤ 0.01), however in test weeks 10 to 12 no significant changes were noted for the relative food consumption. Accordingly, high-dosed female animals showed slightly lower body weights of up to 10.7% (statistically significant at p ≤ 0.01) in test weeks 10 to 12. All changes are within the normal range of biological variation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
The female animals treated with the intermediate dose of 300 mg act. ingr./kg bw/day revealed a slight increase of the drinking water consumption by up to 25.9% (in test week 13) compared to the control group being statistically significant (at p ≤ 0.05 or p ≤ 0.01) in test weeks 1 to 8, 11, and 13.
At the high dose of 1000 mg act. ingr./kg bw/day, both the male and the female animals revealed an increase of the drinking water consumption by up to 64.0% (in test week 4) for the male animals and by up to 60.6% (in test week 3) for the female animals in comparison to the control group as of test week 1. The effect was statistically significant (at p ≤ 0.05 or p ≤ 0.01) in test weeks 1 to 13 for the male and female animals and appeared to be slightly more pronounced in the female animals than in the male animals. . The increased drinking water consumption is considered a test item-related effect.
Recovery period: No noteworthy difference was noted for the drinking water consumption of the male animals previously treated with the high dose in comparison to the control group during the recovery period.
The drinking water consumption of the female animals previously treated with the high dose was still slightly increased by 17.5% (statistically significant at p ≤ 0.05) compared to the control group in test week 14. Afterwards, there was no noteworthy difference between the previously high-dosed animals and the control group.

Ophthalmological findings:
no effects observed
Description (incidence and severity):
Treatment period: No test item-related changes were noted.
Recovery period: No test item-related changes were noted.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period: No test item-related changes were noted in haematology and coagulation.
Recovery period: No test item-related changes were noted.
Some statistically significant differences in haematological parameters were noted in comparison to the control group that are not considered to be test item-related but to be coincidental.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period: No test item-related changes were noted for clinical biochemistry and serum levels of triiodothyronine) (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH).
Recovery period: No test item-related changes were noted.
Some statistically significant differences in biochemical parameters were noted in comparison to the control group that are not considered to be test item-related but to be coincidental.
The serum levels of T4 appeared to be decreased by 24.4% or 25.5% in the male animals treated with 300 or 1000 mg act. ingr./kg bw/day (statistically significant at p ≤ 0.01 for both groups) in comparison to the control animals at the end of the treatment period, respectively (test day 91, only the main study animals). The female animals revealed a decrease by 22.4% (not statistically significant at p ≤ 0.05 or p ≤ 0.01) at 300 mg act. ingr./kg bw/day but an increase by 8.7% at 1000 mg act. ingr./kg bw/day in comparison to the control (also not statistically significant).
The mode of toxicological action on the thyroid gland by various substances is quite similar, independently of the mechanism that leads to the reduction of the T4 level. Increased release of TSH from the pituitary gland is observed as a response to a decreased circulating T4 level. However, no influence was noted on the thyroid weight, and no histopathological findings were observed for the thyroids in animals treated with the test item. Therefore, a toxicological relevance of the decreased T4 serum levels cannot be confirmed for the test item due to the absence of any of the described changes expected to follow a toxicity-related decrease in T4 serum levels. Hence, these changes are considered coincidental alterations within the range of normal biological variability, as only 1 animal in the intermediate and high dose group each showed T4 serum levels outside the historical range.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period: No test item-related changes were noted for urinalysis.
Recovery period: No test item-related changes were noted.
A large amount ('+++') of erythrocytes in the centrifugal deposits and an increased haemoglobin content (250 erythrocytes/µL) were observed in the urine of 4 of 10 male animals (nos. 71, 72, 75, and 77) treated with 1000 mg act. ingr./kg bw/day at the end of the treatment period and one male control animal. For all animals, all findings observed (increased number of erythrocytes, increased haemoglobin content, urine colour) are not regarded to be test item-related effects. For animals no. 71, 72 and 75 as well as the single control animal, we consider these changes most likely to be due to small injuries at the animals' claws caused by the metal grid of the funnel cages that might not have been detected at necropsy. For animal no. 77, there are two possible causes for the findings. However, due to animal no. 77 being the only high dose animal to show macroscopic and microscopic changes in the related organs, we consider these to be spontaneous and not test item-related.
Some other statistically significant differences were noted for the urinary parameters in comparison to the control group that are not considered to be test item-related but to be coincidental.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related and/or statistically significant influence was noted on any of the parameters examined during the functional observation tests, on the fore- and hindlimb grip strength, or on the spontaneous motility for any of the male and female animals after repeated oral treatment with 100, 300, or 1000 mg act. ingr./kg bw/day in test week 13 and in test week 17. In female animals treated with either 100, 300 or 1000 mg act. ingr./kg bw/day, a decrease in spontaneous motility was noted compared to control animals. This difference was even more pronounced at the end of the recovery period for females previously treated with 1000 mg act. ingr./kg bw/day. However, during the daily observation of the animals no corresponding clinical signs were noted, and the neurological screening revealed no differences in behaviour between the treated and control animals. Therefore, these apparent effects observed in a single test are considered to be spontaneous and not related to any toxic effect of the test item.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period: No test item-related changes were noted in organ weights.
Recovery period: No test item-related changes were noted.
Any differences noted for the relative or absolute organ weights of the test item-treated animals at the end of the treatment period or at the end of the recovery period in comparison to the control animals are not considered to be test item-related and to be within the normal range of biological variation. The increased relative brain weight noted in females treated with 300 or 1000 mg act. ingr./kg bw/day is considered a secondary effect to the reduced body weight at necropsy, as the absolute brain weight does not differ between the control group animals and the animals treated with the test item.
Statistically significant differences in organ weights compared to the control animals that are not considered to be test item-related are listed.
Any differences noted for the relative or absolute organ weights of the test item-treated animals at the end of the treatment period or at the end of the recovery period in comparison to the control animals are not considered to be test item-related and to be within the normal range of biological variation. The increased relative brain weight noted in females treated with 300 or 1000 mg act. ingr./kg bw/day is considered a secondary effect to the reduced body weight at necropsy, as the absolute brain weight does not differ between the control group animals and the animals treated with the test item.
Some statistically significant differences in organ weights compared to the control animals were not considered to be test item-related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment period: One of 10 female animals treated with 1000 mg act. ingr./kg bw/day revealed test item-related changes in form of a thickened mucosa in the cardia region of the stomach with multiple elevated foci with a diameter of 1 to 3 mm that were partly crater-like. Histopathological correlates were noted at microscopic examination.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment period: Histopathology was restricted to control/high dose group, and stomachs of low and intermediate dose groups. Test item-related local changes were noted in the stomach (non-glandular) of almost all animals treated with 1000 mg act. ingr./kg bw/day in form of squamous cell hyperplasia and hyperkeratosis (10 of 10 males, 9 of 10 females) and submucosal mixed inflammatory cell infiltrate (4 of 10 animals per each sex). No local changes were noted in the stomachs at 100 and 300 mg act. ingr./kg bw/day
Recovery period: The histomorphological examination did not reveal any findings related to the previous test item-treatment, in particular no abnormalities were noted in the stomach.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
AUDITORY EXAMINATIONS:
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4): There was no indication of any impairment to auditory acuity.

OESTRUS CYCLE:
-The oestrus cycle stages (proestrous, oestrous, metoestrous, dioestrous) of all main study and recovery females was determined by analysis of vaginal smears taken at necropsy at terminal sacrifice (test day 91) or recovery sacrifice (test day 119). All groups showed a normal variation regarding the frequency of the different oestrus cycle stages.
Details on results:
CLINICAL SIGNS AND MORTALITY
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4):
No test item-related changes in behaviour, external appearance, or consistency of faeces were observed for the male and female rats following repeated oral treatment with 100, 300, or 1000 mg act. ingr./kg bw/day for 90 days.
No signs of irritation were observed following the oral dosing, in particular no salivation or redness of the oral cavity.
None of the animals died prematurely, all animals survived until their scheduled sacrifice at terminal sacrifice or recovery sacrifice.
Detailed clinical observations in form of an assessment of external appearance, body posture, movement and coordination capabilities, as well as behaviour were performed for all animals pre- and post-dose on test day 1 (= TW 1), and always on the first day of the week in test weeks 2 to 13. The observations were made within approx. 2 hours after dosing.
All parameters of the detailed clinical observations of all animals scheduled for the control group or the treatment groups were in the normal range at pre-dose examination on test day 1.
-Treatment period:
None of the animals treated with 100, 300, or 1000 mg act. ingr./kg bw/day revealed any changes in external appearance, body posture, movement and coordination capabilities in test weeks 1 to 13. All male and female control animals revealed normal values for each parameter set examined throughout the treatment period.
The detailed clinical observations were discontinued for the animals scheduled for the recovery period after test week 13 as none of the animals had ever revealed any abnormalities during the treatment period.

BODY WEIGHT AND WEIGHT GAIN
-Treatment period:
No test-item related influence was noted on the body weight, the body weight gain and the body weight at autopsy for the male and female animals treated orally with 100 or 300 mg act. ingr./kg bw/day for 90 days in comparison to the control group during the treatment period.
The mean body weight of the female animals treated with 1000 mg act. ingr./kg bw/day was slightly but statistically significantly (at p ≤ 0.05 or p ≤ 0.01, day 43-50 and 64-90) decreased by up to 10.7% in comparison to the control group as of test day 43. Body weight gain appeared to be slightly lower in females dosed at 1000 mg/kg bw.
The body weight of the male high-dosed animals was also slightly reduced by up to 7.4% (on test day 78) in comparison to the control group, but none of the decreases attained statistical significance (at p ≤ 0.05 or p ≤ 0.01). Furthermore, body weight gain appeared to be slightly lower as well.
The body weight at autopsy of male and female animals treated with 1000 mg act. ingr./kg bw/day was reduced accordingly.
This effect is considered to be test item-related.
The low-dosed male animals revealed a marginal decrease of the body weight (by up to 5.8% in comparison to the control group on test day 85), whereas at the intermediate dose the body weight was always nearly identical to that of the control group.
Given these observations, the marginal decreases of the body weight noted for the male and female animals at the intermediate and/or low dose is not considered to be test item-related but to be within the range of normal biological variation.
- Recovery period (restricted to control and high dose, groups 1 and 4):
A recovery of the reduced body weight of the female animals was noted during the course of the recovery period as the difference to the control group (statistically significant on test days 97 (-8.7%; p ≤ 0.01) and 104 (-7.3%; p ≤ 0.05)) declined to be only -4.5% at study termination (not statistically significant).
The slight difference in body weight noted between the male high-dosed animals and the control animals at the end of the treatment period also declined during the recovery period such that the body weight of the previously high-dosed animals and the control animals was nearly identical at study termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4):
No test-item related influence was noted on the absolute and relative food consumption for the animals treated orally with 100, 300, or 1000 mg act. ingr./kg bw/day for 90 days in comparison to the control group throughout the treatment and recovery period.
For the male high-dosed animals, a slight but statistically significant (at p ≤ 0.05 or p ≤ 0.01) increase by up to 14.4% was noted for the relative food consumption compared to the control animals starting in test week 2 during the treatment period and lasting until test week 16 during the recovery period, however no significant changes were seen in the absolute food consumption. The changes seen for the relative food consumption are within the normal range of biological variation.
In test week 17 (end of recovery), when the previously high-dosed male animals revealed no evident difference in the body weight compared to the control animals, there was also no noteworthy difference in the food consumption between the two groups.
For the female animals treated with 1000 mg act. ingr./kg bw/day, a slight but statistically significant (at p ≤ 0.01) decrease in absolute food consumption of up to 14.6% was noted in test weeks 1 and 10 to 12. In test week 1, the relative food consumption was also slightly decreased (statistically significant at p ≤ 0.01), however in test weeks 10 to 12 no significant changes were noted for the relative food consumption. Accordingly, high-dosed female animals showed slightly lower body weights of up to 10.7% (statistically significant at p ≤ 0.01) in test weeks 10 to 12. In test week 1, no significant differences in bodyweight between female high-dosed and control animals were noted, however the decrease in absolute and relative food consumption is considered spontaneous and not test-item related due to its transient nature. Furthermore, all changes are within the normal range of biological variation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
-Treatment period:
No test-item related influence was noted on the drinking water consumption for the male and female animals treated orally with 100 mg act. ingr./kg bw/day and for the male animals treated with 300 mg act. ingr./kg bw/day for 90 days in comparison to the control group throughout the treatment and recovery period.
The female animals treated with the intermediate dose of 300 mg act. ingr./kg bw/day revealed a slight increase of the drinking water consumption by up to 25.9% (in test week 13) compared to the control group being statistically significant (at p ≤ 0.05 or p ≤ 0.01) in test weeks 1 to 8, 11, and 13.
At the high dose of 1000 mg act. ingr./kg bw/day, both the male and the female animals revealed an increase of the drinking water consumption by up to 64.0% (in test week 4) for the male animals and by up to 60.6% (in test week 3) for the female animals in comparison to the control group as of test week 1. The effect was statistically significant (at p ≤ 0.05 or p ≤ 0.01) in test weeks 1 to 13 for the male and female animals and appeared to be slightly more pronounced in the female animals than in the male animals. Visual appraisal did not reveal any noteworthy water loss due to spilling.
The increase in the drinking water consumption noted for the females at the intermediate dose level and for the males and females at the high dose level is considered to be test item-related and regarded as an adverse finding. No increase in urinary volume was observed in animals of the intermediate and high dose groups, however, this parameter was determined only once at the end of the treatment period. Therefore, changes in urinary volumes as expected following an increase in drinking water consumption might have occurred during the study without being detected.
-Recovery period (restricted to control and high dose - groups 1 and 4):
No noteworthy difference was noted for the drinking water consumption of the male animals previously treated with the high dose in comparison to the control group during the recovery period.
The drinking water consumption of the female animals previously treated with the high dose was still slightly increased by 17.5% (statistically significant at p ≤ 0.05) compared to the control group in test week 14. Afterwards, there was no noteworthy difference between the previously high-dosed animals and the control group.

OPHTHALMOSCOPIC EXAMINATION
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4):
Ophthalmological examination did not reveal any changes of the eyes and the optic region in the animals treated orally with 100, 300, or 1000 mg act. ingr./kg bw/day for 90 days at the end of the treatment period.
No changes of the eyes and the optic region were noted for the animals previously treated with 1000 mg act. ingr./kg bw/day for 90 days at the end of the recovery period.

HAEMATOLOGY
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4):
No test item-related influence was observed on the haematological parameters for the male and female animals treated orally with 100, 300, or 1000 mg act. ingr./kg bw/day compared to the control animals at the end of the treatment period (test day 91, only the main study animals) and at the end of the recovery period (test day 119, groups 1 and 4 only).
No test item-related effects were observed for the haemoglobin content (HGB), the numbers of erythrocytes (RBC), leucocytes (WBC) and platelets (PLT), the relative reticulocyte count (Reti), the haematocrit value (HCT), the relative and absolute differential blood count, the prothrombin time (PT), the activated partial thromboplastin time (aPTT), the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH), and the mean corpuscular haemoglobin concentration (MCHC) at the end of the treatment period and at the end of the recovery period. All data were within the limits of normal biological variability.

CLINICAL CHEMISTRY
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4):
No test item-related influence was observed on the biochemical parameters for the male and female animals treated orally with 100, 300, or 1000 mg act. ingr./kg bw/day compared to the control animals at the end of the treatment period (test day 91, only the main study animals) and at the end of the recovery period (test day 119, groups 1 and 4 only).
No test item-related effects were noted on the plasma levels of albumin, bile acids, bilirubin, total cholesterol, HDL cholesterol, LDL cholesterol, creatinine, glucose, total protein, urea (in blood), calcium, chloride, potassium, and sodium at the end of the treatment period and at the end of the recovery period. No test item-related influence was noted on the plasma enzyme activities of alanine amino-transferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT), and lactate dehydrogenase (LDH). All data are within the range of normal biological variability.
No test item-related influence was observed on the thyroid hormone levels for the male and female animals following daily oral treatment with 100, 300, or 1000 mg act. ingr./kg bw/day compared to the control animals at the end of the treatment period (test day 91, only the main study animals) and at the end of the recovery period (test day 119, groups 1 and 4 only).
No test item-related effects were noted on the serum levels of T3 (thyroid hormones triiodothyronine), T4 (thyroxine), TSH (thyroid-stimulating hormone). All data are considered to be within the range of normal biological variability.
The serum levels of T4 appeared to be decreased by 24.4% or 25.5% in the male animals treated with 300 or 1000 mg act. ingr./kg bw/day (statistically significant at p ≤ 0.01 for both groups) in comparison to the control animals at the end of the treatment period, respectively (test day 91, only the main study animals). The female animals revealed a decrease by 22.4% (not statistically significant at p ≤ 0.05 or p ≤ 0.01) at 300 mg act. ingr./kg bw/day but an increase by 8.7% at 1000 mg act. ingr./kg bw/day in comparison to the control (also not statistically significant).
The mode of toxicological action on the thyroid gland by various substances is quite similar, independently of the mechanism that leads to the reduction of the T4 level. Increased release of TSH from the pituitary gland is observed as a response to a decreased circulating T4 level. However, no influence was noted on the thyroid weight, and no histopathological findings were observed for the thyroids in animals treated with the test item. Therefore, a toxicological relevance of the decreased T4 serum levels cannot be confirmed for the test item due to the absence of any of the described changes expected to follow a toxicity-related decrease in T4 serum levels. Hence, these changes are considered coincidental alterations within the range of normal biological variability, as only 1 animal in the intermediate and high dose group each showed T4 serum levels outside the historical range.
It is generally known that a histopathological examination of the thyroid is usually more sensitive and thus more reliable in detecting thyroid-related toxic effects of a test item than thyroid hormone levels and thyroid weight. According to the validation report of OECD Guideline 407 (referenced in OECD Guideline 408) 'thyroid histopathology was consistently the most reliable and most sensitive endpoint for the detection of thyroid modulation. Thyroid weight was reliable, but was somewhat less sensitive when compared to thyroid histopathology. Circulating thyroid hormone levels (T3, T4, and TSH) were not always reliable and sensitive, but the standard operating procedures for blood sampling and for thyroid hormone analyses were not standardised to reduce stress induced variability and to reduce analytical variability, respectively. Circulating T4 levels were the most promising of the three thyroid hormonal values'.

URINALYSIS
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4):
Daily oral treatment with 100, 300, or 1000 mg act. ingr./kg bw/day did not lead to any test item-related changes of the urinary parameters in the male and female animals compared to the control group at the end of the treatment period (test day 91, only the main study animals) and at the end of the recovery period (test day 119, groups 1 and 4 only).
No test item-related changes were noted for the specific gravity, the pH value of the urine and the urine volume. The analyte concentrations of nitrite, protein, glucose, ketones, urobilinogen, bilirubin, and haemoglobin were not influenced in a test item-related way in male and female animals. No test item-related changes were observed in the urine colour and the microscopically analysed urine sediments.
A large amount ('+++') of erythrocytes in the centrifugal deposits and an increased haemoglobin content (250 erythrocytes/µL) were observed in the urine of 4 of 10 male animals (nos. 71, 72, 75, and 77) treated with 1000 mg act. ingr./kg bw/day at the end of the treatment period. The colour of these animals' urine was brown (no. 71), brown-yellow (nos. 72 and 75), or red (no. 77). For animal no. 77, a small injury at one claw was noticed at the end of the urine collection, which might have been a source for blood contaminating the urine sample. However, during necropsy of this animal several findings were noted (i.e. urinary bladder filled with red liquid, dilated urether, and enlarged kidneys) that also might have been the cause for the erythrocytes found in the urine sample. For the kidney and urether, respective findings were observed at histopathological examination of these organs. For animal no. 71, a minimal dilation of the pelvis of one kidney was noted during histopathological examination, however no corresponding macroscopic changes were noted during necropsy for animal no. 71, and neither macroscopic nor microscopic changes were noted in animals no. 72 and 75. In addition, no paw injuries were observed in any of these animals. However, the urine was collected overnight without continuous observation of the animals. Any small injuries which may have occurred would have been licked clean by the animals by morning, so that these injuries would not have been detected by our staff. Hence, the source of the blood contamination in the urine samples of animals no. 71, 72 and 75 cannot be determined and there is no data to either support or disaprove a causal relationship with the test item treatment.
The same findings as noted for the 4 high-dosed animals were also observed for one male control animal (no. 10, urine colour: brown). For this animal, this finding has not to be considered incidental as no test item was administered to this animal.

NEUROBEHAVIOUR
The neurological screening was performed on all main study and recovery animals (groups 1 and 4: n = 15 per group per sex, groups 2 and 3: n = 10 per group and sex) at the end of treatment (in test week 13) 1 to 2 hours after dosing, and on all recovery animals (groups 1 and 4: n = 5 per group and sex) at the end of the recovery period (in test week 17).
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4):
No test item-related and/or statistically significant influence was noted on any of the parameters examined during the functional observation tests, on the fore- and hindlimb grip strength, or on the spontaneous motility for any of the male and female animals after repeated oral treatment with 100, 300, or 1000 mg act. ingr./kg bw/day in test week 13 and in test week 17. In female animals treated with either 100, 300 or 1000 mg act. ingr./kg bw/day, a decrease in spontaneous motility was noted compared to control animals. This difference was even more pronounced at the end of the recovery period for females previously treated with 1000 mg act. ingr./kg bw/day. However, during the daily observation of the animals no corresponding clinical signs were noted, and the neurological screening revealed no differences in behaviour between the treated and control animals. Therefore, these apparent effects observed in a single test are considered to be spontaneous and not related to any toxic effect of the test item.
For all animals, all findings observed (increased number of erythrocytes, increased haemoglobin content, urine colour) are not regarded to be test item-related effects. For animals no. 71, 72 and 75 as well as the single control animal, we consider these changes most likely to be due to small injuries at the animals' claws caused by the metal grid of the funnel cages that might not have been detected at necropsy. For animal no. 77, there are two possible causes for the findings. However, due to animal no. 77 being the only high dose animal to show macroscopic and microscopic changes in the related organs, we consider these to be spontaneous and not test item-related.

ORGAN WEIGHTS
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4):
No test-item related influence was noted on the relative and absolute organ weights of the male and female animals following repeated oral treatment with 100, 300 or 1000 mg act. ingr./kg bw/day in comparison to the control animals at the end of the treatment period (test day 91, only the main study animals) and at the end of the recovery period (test day 119, groups 1 and 4 only).
Any differences noted for the relative or absolute organ weights of the test item-treated animals at the end of the treatment period or at the end of the recovery period in comparison to the control animals are not considered to be test item-related and to be within the normal range of biological variation. The increased relative brain weight noted in females treated with 300 or 1000 mg act. ingr./kg bw/day is considered a secondary effect to the reduced body weight at necropsy, as the absolute brain weight does not differ between the control group animals and the animals treated with the test item.

GROSS PATHOLOGY
-Treatment period:
The macroscopic inspection at necropsy did not reveal any test item-related changes in the organs and tissues of the male and female animals repeatedly treated orally with 100 or 300 mg act. ingr./kg bw/day at terminal sacrifice at the end of the treatment period (test day 91).
One of 10 male high dose animals (no. 77) revealed enlarged kidneys, bilateral dilated urethers and a urinary bladder filled with red liquid that are not considered test item-related. The changes in the kidneys and urethers correlated with histopathological findings.
One of 10 female high dose animals (no. 87) revealed a thickened mucosa in the cardia region of the stomach with multiple elevated foci with a diameter of 1 to 3 mm that were partly crater-like. These changes are considered to be test item-related as histopathological correlates were noted at microscopic examination.
-Recovery period (restricted to control and high dose - groups 1 and 4):
No test item-related changes were noted for the male and female animals previously treated with 1000 mg act. ingr./kg bw/day at the end of the recovery period.
A few minor macroscopic findings were noted in various organs of individual animals at terminal sacrifice or recovery sacrifice. These changes are not regarded as test item-related but to be of spontaneous nature due to their isolated occurrences. In detail, these findings were:
Enlarged kidneys, increased lobular pattern of the liver, slightly enlarged spleen, enlarged thyroids, dilated urether, urinary bladder filled with red liquid, dilated uterus, or uterus filled with clear liquid.

HISTOPATHOLOGY: NON-NEOPLASTIC
The histological examination was restricted to the main study and recovery animals of group 1 (control) and group 4 (high dose) and was performed by Global Pathology Support, The Netherlands. In order to further assess test item-related stomach changes observed for both sexes in selected group 4 animals, further histopathological evaluation of the stomachs of group 2 and 3 (low/intermediate dose) was performed in both sexes.
Histopathology evaluation of group 4 revealed test item-related changes in the non-glandular part of the stomach (i.e. the forestomach) in almost all animals for squamous cell hyperplasia and hyperkeratosis (10 of 10 males and 9 of 10 females). Furthermore, submucosal mixed inflammatory cell infiltrate was observed in the non-glandular and the glandular part of the stomach (4 of 10 animals each per sex).
Macroscopic changes noted in form of a thickened mucosa in the cardiac region of the stomach with multiple elevations for one female animal (no. 87) treated with 1000 mg act. ingr./kg bw/day were associated with the microscopic findings of squamous cell hyperplasia and hyperkeratosis of the non-glandular stomach, and mixed inflammatory cell infiltrate in the submucosa (glandular and non-glandular part).
The histopathological evaluation of the stomachs of group 2 and 3 (low/intermediate dose) did not reveal any test item-related effects.
The animals from group 4 scheduled for the 28-day recovery did not show any test item-related findings at the end of the recovery period.

OTHER FINDINGS
AUDITORY EXAMINATIONS:
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4):
There was no indication of any impairment to auditory acuity.

OESTRUS CYCLE:
The oestrus cycle stages (proestrous, oestrous, metoestrous, dioestrous) of all main study and recovery females was determined by analysis of vaginal smears taken at necropsy at terminal sacrifice (test day 91) or recovery sacrifice (test day 119). All groups showed a normal variation regarding the frequency of the different oestrus cycle stages.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Remarks:
Read-across source test item
Sex:
male/female
Basis for effect level:
body weight and weight gain
water consumption and compound intake
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Validation report: Validation of an analytical method for the determination of Butanedioic acid, 2 (or 3)-sulfo-, 4-[2-[(1-oxo-(C12 -C18 (even numbered) and C18 unsaturated)alkyl))amino]ethyl]esters disodium salts in test item formulations with subsequent HPLC-UV detection. LPT Study No. 36867.

The aim of this study was to validate a HPLC-UV method for the quantification of Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18 (even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts (also referred to as 'act.ingr.') in tap water formulations.

The analytical method applied was validated by LPT with regard to the linearity of the calibration curve as well as accuracy, precision, stability, specificity and sensitivity. The validation results are summarised in the table below.

Table 1. Summary of validation results

Parameter

Method for the determination of Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12- C18 (even numbered) and C18 unsaturated)alkyl))amino]ethyl]esters, disodium salts in test item formulation samples.

Linearity

R2 = 0.9999 (concentration range: 100 to 1000μg/mL)

Accuracy

Inaccuracy = 0.64% (mean)

Precision

Imprecision

Imprecision ≤ 1.04% CV intra-day

Imprecision = 0.68% CV inter-day

Stability

No apparent degradation of processed samples after 24h storage in the autosampler.

No apparent degradation in tap water after 42 days of storage at -20°C.

 

Sensitivity

(calculated)

Lower Limit of Quantification (LLOQ) = 24.55μg/mL

Limit of Detection (LOD) = 8.10μg/mL

Specificity

No peak interferences at the retention time of Butanedioic acid, 2(or 3)-sulfo-, 4-[2 -[(1-oxo(C12-C18 (even numbered) and C18 unsaturated)alkyl))amino]ethyl]esters, disodium salts in formulation samples at the applied concentrations.

 

The results of the validation confirm that the method employed is suitable for the determination and quantification of Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18 (even numbered) and C18 unsaturated)alkyl))amino]ethyl]esters, disodium salts in tap water formulations.

Conclusions:
Under consideration of the observations described above, in particular the reduced body weight and the substantially increased water consumption, the experimental no-observed-adverse-effect level (NOAEL) for the read-across test item was 300 mg act. ingr./kg bw by daily oral administration.
None of the animals previously treated with 1000 mg act. ingr./kg bw/day revealed any abnormalities related to the previous treatment during the recovery period, thus all toxicological relevant changes observed at the end of the treatment period were completely reversible within the 4-week recovery period.
Executive summary:

The aim of the study was to obtain information on the toxicity of read-across test item Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18 (even numbered) and C18 unsaturated)alkyl)) amino]ethyl]esters, disodium salts (= 'act. ingr.') at dose levels of 100, 300, or 1000 mg/kg bw given to rats by daily oral administration via gavage for 90 days, and the reversibility of any effects after a 4-week treatment-free recovery period.

 

Treatment period:

No deaths were noted at any dose level. All animals survived until their scheduled sacrifice.

None of the male and female rats treated orally with 100, 300, or 1000 mg act. ingr./kg bw/day revealed any test item-related changes in behaviour, external appearance, consistency of faeces, body posture, and movement and coordination capabilities

No test item-related effects were noted during the observational screening and the functional tests.

The mean body weight of the male and female animals treated with 1000 mg act. ingr./kg bw/day was slightly reduced by up to 10.7% in females and up to 7.4% in males in comparison to the control group.

The body weight gain and the body weight at autopsy of both sexes were reduced accordingly. This effect is considered to be test item-related.

No toxicologically relevant test item-related influence was noted on absolute and relative food consumption

Treatment with 300 mg act. ingr./kg bw/day led to an increase of the drinking water consumption of the female animals by up to 25.9% in comparison to the control group starting in test week 1. No effect was noted for the corresponding male animals. At the high dose of 1000 mg act. ingr./kg bw/day, both the male and the female animals revealed an increase of the drinking water consumption by up to 64.0% in comparison to the control group starting in test week 1. The increased drinking water consumption is considered a test item-related effect.

No test item-related changes were noted in haematological and coagulation parameters.

No test item-related changes were noted in clinical chemistry.

No test item-related or toxicologically relevant influence was noted on the serum levels of triiodothyronine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH).

No test item-related changes were noted in urinalysis.

No test item-related changes were noted in ophtalmological and auditory examination.

At necropsy, one of 10 female animals treated with 1000 mg act. ingr./kg bw/day revealed test item-related changes in form of a thickened mucosa in the cardia region of the stomach with multiple elevated foci with a diameter of 1 to 3 mm that were partly crater-like. Histopathological correlates were noted at microscopic examination.

No test item-related changes were noted in organ weights.

Histopathological examination restricted to control and high dose group, revealed test item-related local changes in the stomach (non-glandular) of almost all animals treated with 1000 mg act. ingr./kg bw/day in form of squamous cell hyperplasia and hyperkeratosis (10 of 10 males, 9 of 10 females) and submucosal mixed inflammatory cell infiltrate (4 of 10 animals per each sex).

Histopathological stomach examination of low and intermediate dose groups revealed no local changes in the stomachs at 100 and 300 mg act. ingr./kg bw/day.

Recovery period:

None of the animals previously treated with 1000 mg act. ingr./kg bw/day revealed any abnormalities related to the previous treatment during the 4-week recovery period.

The body weight of the male and female animals recovered towards the normal range, no noteworthy difference compared to the control group was noted at the end of the recovery period.

The drinking water intake of all previously high-dosed animals was in the normal range during the recovery period.

The histomorphological examination did not reveal any findings related to the previous test item-treatment, in particular no abnormalities were noted in the stomach.

Under consideration of the observations described above, in particular the reduced body weight and the substantially increased water consumption, the experimental NOAEL for the read-across test item was 300 mg act. ingr./kg bw by daily oral administration.

None of the animals previously treated with 1000 mg/kg bw/day revealed any abnormalities related to the previous treatment during the recovery period, thus all toxicological relevant changes observed at the end of the treatment period were completely reversible within the 4 -week recovery period.

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
High quality (Klimisch 1)
Organ:
stomach

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

In the absence of any evidence for species specific effects or modes of action (beside changes in the forestomach) the effects observed in animals and the absence of effects are regarded as relevant for humans.

Additional information

Data were available for read across substance EC 939 -637 -2 or 'Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts':

- In a supporting 14-day dose-range-finding study the dose levels were selected for a combined repeated dose and reproduction/developmental toxicity screening test (Hansen, 2013a). 5 Male and 5 female rats were treated once daily with a liquid formulation containing 41.5% active ingredient at dose levels of 100, 300 and 1000 mg act. ingr./kg bw/day by oral gavage administration. None of the animals died prematurely. Salivation was noted for 2 of 5 male animals treated with 1000 mg/kg bw/day starting on day 9 and increased faeces was noted for 3 of 5 male and 2 of 5 female high dosed animals starting on test day 5. The food consumption of the male and female animals treated with 1000 mg/kg bw/day was slightly increased by 9% for the males and by 10% for the females in test week 2. None of the male and female rats treated orally with 100, 300 or 1000 mg/kg bw/day revealed any test item-related changes in body weight, body weight gain as well as relative and absolute organ weights or at macroscopic inspection at necropsy. NOAEL was 300 mg/kg bw/day.

- A key study for repeated dose toxicity was performed by means of an oral combined repeated dose and reproduction/development screening study according to OECD guideline 422 (Hansen, 2013b). The test item was administered orally by gavage to rats with a liquid formulation containing 41.5% active ingredient at dose levels of 100, 300 and 1000 mg/kg bw/day for at least 28 days in male rats and at least 39 days in females. No test item-related premature death was noted in any treatment group. No signs of clinical toxicity were noted for the male and female rats of the low and intermediate dose groups (100 and 300 mg/kg bw/day), whereas slightly increased salivation was noted in one male rat at 1000 mg/kg bw/day. No observational and functional neurological findings were seen up to the highest dose group. A slight reduction in body weight was noted for the male and female rats dosed at 1000 mg/kg bw/day. The laboratory examinations revealed an increased serum ALAT activity for the male and female rats dosed at 1000 mg/kg bw/day, and a decreased serum albumin concentration for the male rats of the high dose group. No test item-related changes were noted during the macroscopic inspection at autopsy with the exception of changes in the stomach from 2 male animals from the high dose group. Microscopic examination revealed the occurrence of squamous cell hyperplasia in the non-glandular mucosa of the forestomach and acute inflammation in the male and female rats of the high dose group. These changes are considered to be local, and not relevant for humans as humans lack a forestomach. NOAEL for paternal/maternal toxicity was 300 mg/kg bw/day.

- A key 90-day repeated dose toxicity study was conducted at dose levels of 100, 300, or 1000 mg/kg bw given to rats by daily oral administration via gavage for 90 days, and the reversibility of any effects after a 4-week treatment-free recovery period was studied (Leuschner, 2020).

After the 90-day treatment period, no deaths were noted at any dose level. None of the male and female rats treated orally with 100, 300, or 1000 mg act. ingr./kg bw/day revealed any test item-related changes in behaviour, external appearance, consistency of faeces, body posture, and movement and coordination capabilities. No test item-related effects were noted during the observational screening and the functional tests.

The mean body weight of the male and female animals treated with 1000 mg act. ingr./kg bw/day was slightly reduced by up to 10.7% in females and up to 7.4% in males in comparison to the control group. The body weight gain and the body weight at autopsy of both sexes were reduced accordingly. This effect is considered to be test item-related.

No toxicologically relevant test item-related influence was noted on absolute and relative food consumption. Treatment with 300 mg act. ingr./kg bw/day led to an increase of the drinking water consumption of the female animals by up to 25.9% in comparison to the control group starting in test week 1. No effect was noted for the corresponding male animals. At the high dose of 1000 mg act. ingr./kg bw/day, both the male and the female animals revealed an increase of the drinking water consumption by up to 64.0% in comparison to the control group starting in test week 1. The increased drinking water consumption is considered a test item-related effect.

No test item-related changes were noted in haematological and coagulation parameters, clinical chemistry, serum levels of triiodothyronine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH) and urinalysis.

No test item-related changes were noted in ophtalmological and auditory examination.

At necropsy, one of 10 female animals treated with 1000 mg act. ingr./kg bw/day revealed test item-related changes in form of a thickened mucosa in the cardia region of the stomach with multiple elevated foci with a diameter of 1 to 3 mm that were partly crater-like. Histopathological correlates were noted at microscopic examination.

No test item-related changes were noted in organ weights.

Histopathological examination restricted to control and high dose group, revealed test item-related local changes in the stomach (non-glandular) of almost all animals treated with 1000 mg act. ingr./kg bw/day in form of squamous cell hyperplasia and hyperkeratosis (10 of 10 males, 9 of 10 females) and submucosal mixed inflammatory cell infiltrate (4 of 10 animals per each sex). Histopathological stomach examination of low and intermediate dose groups revealed no local changes in the stomachs at 100 and 300 mg act. ingr./kg bw/day.

After the recovery period, none of the animals previously treated with 1000 mg act. ingr./kg bw/day revealed any abnormalities related to the previous treatment during the 4-week recovery period. The body weight of the male and female animals recovered towards the normal range, no noteworthy difference compared to the control group was noted at the end of the recovery period. The drinking water intake of all previously high-dosed animals was in the normal range during the recovery period. The histomorphological examination did not reveal any findings related to the previous test item-treatment, in particular no abnormalities were noted in the stomach.

Under consideration of the observations described above, in particular the reduced body weight and the substantially increased water consumption, the experimental NOAEL was 300 mg act. ingr./kg bw by daily oral administration.

 

Conclusion

The NOAEL of 300 mg/kg bw in the OECD 422 study with 'Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts' was considered as the most reliable and conservative value, therefore this was selected as the descriptor for DNEL calculations (justification with data matrix separately attached in Section 13).

Justification for classification or non-classification

Based on these results and according to the CLP (No. 1272/2008 of 16 December 2008), the test substance does not have to be classified and has no obligatory labelling requirement for repeated dose toxicity.