Butanedioic acid, sulfo-, 4-[2-[(2-hydroxyethyl)amino]ethyl] ester, N-C18-unsatd. acyl derivs., disodium salts

Endpoint summary

Description of key information

Key data for subacute toxicity were available from an oral (gavage) OECD 422 study in rats with read-across substance '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 were observed as systemic changes, whereas macroscopic and microscopic stomach changes were observed as local changes, the latter without relevance to humans. NOAEL for paternal/maternal toxicity was 300 mg/kg bw/dayy.
Key data for subchronic toxicity were available from an oral (diet) 90-day toxicity study in rats with read-aross substance 'Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium salts' dosed at 0.5, 2 and 8 (reduced to 4) g/kg bw.day. The study showed decreased body weight gain, feed consumption and food efficiency at the mid dose and high dose levels and increased SGOT and SGPT (liver enzymes) at the high dose. Further hematuria was seen in the mid and high dose rats, various organ weights were decreased (e.g. decrease in adrenal and gonadal weight in high dose groups; decrease in pituitary weight in females of high dose group) and lower urinary tract pathology was seen in 2 high dosed rats. It thus appeared that the NOEL in the rat was below 0.50 g/kg/day, however 0.5 g act.ingr./kg bw/day can be considered as NOAEL. 
The NOAEL of 300 mg/kg bw in the OECD 422 study was considered as the most reliable and conservative NOAEL for risk characterisation. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2012-2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to GLP and valid methods and is considered relevant and reliable for classification.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: CD® / Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Research, Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at start of dosing: Males 55 days; Females: 48 days
- Weight range at start of dosing: Males: 281.0 to 308.0 g; Females: 68.9 to 195.7 g
- Fasting period before study: Ad libitum with exception of the night before the day of blood withdrawal for laboratory examination.
- Housing: With exception of the mating period, the animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt/ Arkeburg, Germany) was used as bedding material in these cages. The cages were cleaned and changed once a week.
- Diet (e.g. ad libitum): ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany, ad libitum with exception of the night before the day of blood withdrawal for laboratory examination.
- Water (e.g. ad libitum): Tap water was offered daily ad libitum.
- Acclimation period: 5 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%): 55% ± 15% (maximum range)
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
Males From: August 27, 2012 To: October 2, 2012
Females From: August 27, 2012 To: October 19, 2012

Route of administration:

oral: gavage

Vehicle:

other: tap water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Application volume: 5 mL/kg bw/day. The test item was dissolved in the vehicle tap water to concentrations of 20, 60 and 200 mg test item /mL tap water and was administered orally at a constant volume once daily. The amount of the test item was adjusted to the animal's current body weight daily. The test item-vehicle mixture was freshly prepared every day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the analysis of the test item-vehicle mixtures samples of approx. 2 x 5 mL were taken at the following time points and stored at ≤ -20°C until analysis at LPT.
*Start of treatment period: Analysis of stability and concentration: Immediately after preparation of the test item-vehicle mixtures as well as 8 and 24 hours after storage of the test item preparations at room temperature: 3 samples/dose level group = 9 samples
*End of treatment period: Concentration: During treatment with the test item always before administration to the last animal/dose level group: 3 samples
The samples were labelled with the study number, test item, test species, type of sample, aliquot number, concentration, test day, sampling time and date.
The validation of the analytical method is part of LPT study No. 28344 (14-day dose-range-finding).

The measured actual concentrations of the test item in the test item vehicle mixtures were between 99.99% and 102.96% of the nominal concentrations (table 26).

Duration of treatment / exposure:

Males: 2 weeks prior to mating, during the mating period and approx. 2 weeks post mating and continuing up to test day 36 (one day before sacrifice).
Females:2 weeks prior to mating and continuing up to, and including, day 3 post-partum or the day before sacrifice.

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels have been selected in agreement with the Sponsor based on the results of a 14-day dose-range-finding study in rats dosed at 100, 300 and 1000 mg act. ingr./kg bw by oral gavage (LPT Study No. 28344). None of the animals died prematurely. None of the male and female rats treated orally with 100 or 300 mg act. ingr./kg bw/day revealed any changes in behaviour, external appearance or faeces. Salivation was noted for 2 of 5 male animals treated at 1000 mg/kg bw/day on 1 or 3 test days starting on test day 9 and increased faeces was noted for 3 of 5 male and 2 of 5 female high dosed animals on 6 or 9 test days starting on test day 5. No test item-related changes on body weight and body weight gain were noted for the male and female rats up to 1000 mg act. ingr./kg bw/day. No test item-related changes on food consumption were noted for the male and female rats treated orally with 100 or 300 mg act. ingr./kg bw/day. The food consumption of the male and female animals treated with 1000 mg act. ingr./kg bw/day was slightly increased by 9% for the males and by 10% for the females in test week 2 (statistically significant at p ≤ 0.01 for both sexes). No test item-related influence was noted for the drinking water consumption at any of the tested dose levels. None of the male and female rats treated orally with 100, 300 or 1000 mg act. ingr ./kg bw/day revealed changes at macroscopic inspection at necropsy or organ weights.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the test period, each animal was observed for clinical signs at least once daily. Mortality was recorded twice daily. In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11 :00 a.m. with a final check performed at approximately 3:30 p.m.
- Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Additionally, once before the first exposure (to allow within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena and at the same time, each time preferably by observers unaware of the treatment. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes. Body weights were recorded individually for each adult animal.
- Time schedule for examinations:
Males and females were weighed on the first day of dosing, weekly thereafter and at termination. During gestation, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum) and day 4 post-partum.

FOOD CONSUMPTION: Yes
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period with the execution of the mating period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week.

WATER CONSUMPTION:
- Time schedule for examinations: Water consumption was monitored daily by visual appraisal throughout the study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period
- Anaesthetic used for blood collection: Yes (identity) ether anaesthesia
- Animals fasted: Yes , overnight
- How many animals: 5 male and 5 female animals randomly selected from each group.
- Parameters:
Differential blood count (relative)
Differential blood count (absolute)
Erythrocytes
Leucocytes
Haematocrit value
Haemoglobin content
Platelets
Reticulocytes
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)
Coagulation: Thromboplastin time & Activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the premating period
- Animals fasted: Yes, overnight
- How many animals: 5 male and 5 female animals randomly selected from each group.
- Parameters:
Sodium
Potassium
Calcium
Chloride
Albumin
Globulin
Albumin/globulin ratio
Total bilirubin
Total cholesterol
Creatinine
Glucose
Total protein
Blood urea
Alanine amino- transferase (ALAT)
Alkaline phosphatase (aP)
Aspartate aminotransferase (ASAT)
Bile acids

URINALYSIS: No

OTHER/ REPRODUCTIVE PARAMETERS (See Section 7.8.1 & 7.8.2)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: shortly before sacrifice (males); during lactation also shortly before sacrifice (females)
- Dose groups that were examined: five males and five females randomly selected from each group
- Battery of functions tested:
1. Observational screening
Righting reflex
Body temperature
Salivation
Startle response
Respiration
Mouth breathing
Urination
Convulsions
Pilo-erection
Diarrhoea
Pupil size
Pupil response
Lacrimation
lmpaired gait
Stereotypy
Toepinch
Tail pinch
Wire maneuver
Hind leg splay
Positional passivity
Tremors
Positive geotropism
Limb rotation
Auditory function
2. Functional tests
Grip strenght
Locomotor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- At the time of sacrifice, or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI.
The numbers of corpora lutea and implantation sites were recorded in the female adult animals and reported.
- Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.

ORGAN WEIGHTS: Yes
- The following organs of all adult animals were weighed individually and identified as left or right: Epididymis (2), Testicle (2)
- Determination of the organ weights of the following organs was only performed from 20 adult males and 20 adult females, which were randomly selected: Adrenal gland (2), Heart, Liver, Thymus, Brain, Kidney (2), Spleen, Adrenal glands and kidneys were weighed individually and identified as left or right.
- Animals Nos.
Group 1: 1, 2, 4, 8, 9 12, 13, 14, 18, 20
Group 2: 21, 22, 23, 27, 28 33, 35, 36, 39, 40
Group 3: 44, 45, 46, 47, 49 52, 53, 56, 58,60
Group 4: 61, 62, 63, 67, 69 72, 73, 74, 77, 79

HISTOPATHOLOGY: Yes
- The following organs or parts of organs of all adult animals were fixed in 7% formalin; testes and epididymides were fixed in Bouin's fixative:
Epididymis (2), Gross lesions, Mammary gland, Ovary (2), Prostate, Seminal vesicle, Testicle (2), Uterus (incl. cervix and oviducts), Vagina
-In addition, the following organs or parts of organs of the selected 20 adult males and 20 adult females (see section above) were fixed in 7% formalin:
Adrenal gland (2)
Bone marrow (os femoris)
Brain (cerebrum, cerebellum, brain stem)
Heart (left and right ventricle, septum)
Intestine, small (duodenum, jejunum, ileum,
incl. Peyer's patches, Swiss roll method)
Intestine, large (colon, rectum)
Kidney and ureter (2)
Liver
Lungs (with mainstem bronchi and
bronchioles), preserved by inflation with
fixative and then immersion
Lymph node (1, cervical), Lymph node (1, mesenteric)
Nerve (sciatic)
Oesophagus
Spinal cord (3 sections)
Spleen
Stomach
Thyroid (incl. parathyroids)
Thymus
Tissue masses or tumours (incl.
regional lymph nodes)
Tongue (incl. base)
Trachea (incl. larynx)
Urinary bladder
-Only the 10 selected animals from the control group and the high dose group (20 animals in total) were considered for histopathological evaluation.

Other examinations:

Fertility and reproduction: See section 7.8.1 and 7.8.2
Organ weights: tables 14-1to 14-4 (relative) and 15-1 to 15-4 (absolute)

Statistics:

Toxicology and Pathology data were captured, whenever possible, using the departmental computerized systems (Provantis®8 Integrated preclinical software, Instem LSS Ltd.). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs.
The test item-treated groups (2 - 4) were compared with the control group (1 ).

The following statistical methods were used:

STUDENT' s t-test: All numerical functional tests (p ≤0.01)

Multiple t-test based on DUNNETT, C. W., New tables for multiple comparisons with a control, Biometrics, 482-491 (Sept 1 964): Body weight I Food consumption I Haematology I Clinical chemistry I Absolute and relative organ weights: (p ≤ 0.05 and ≤0.01)

For all numerical values (e.g. body weight, food consumption and organ weight data) homogeneity of variances was tested by using the BARTLETT chi2-test.

lf the variances are homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group.

In case of heterogeneity of variances, the STUDENT's t-test was carried out; limit of significance is p ≤ 0.01.

Exact test of R. A. FISHER: Histopathology, if applicable (p≤0.05)

For the comparison of classification measurements (for example the fertility index) the FISHER's exact test, n < 100 or chi2-test with Yates' correction for continuity, n ≥ 100 (p ≤0.05 and p ≤0.01) were employed.

These statistical procedures were used for all data. Significantly different data were indicated in the tables of the report.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to ± 1 may occur caused by rounding.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

salivation in one male rat at 1000 mg/kg bw/day

Mortality:

mortality observed, treatment-related

Description (incidence):

salivation in one male rat at 1000 mg/kg bw/day

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

slight reduction in male and female rats at 1000 mg/kg bw/day

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

slight increase in food consumption in male rats dosed at 1000 mg/kg bw/day

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

increased ALAT in male and female rats dosed at 1000 mg/kg bw/day; decrease in albumin in male rats dosed at 1000 mg/kg bw/day;

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

changes in the stomach from 2 male animals dosed at 1000 mg/kg bw/day: whitish thickening (cardia), yellowish contents

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

squamous cell hyperplasia and in the non glandular mucosa and acute inflammation in male and female rats dosed at 1000 mg/kg bw/day; pulmonary congestion in male and female rats dosed at 1000 mg/kg bw/day

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No test item-related premature death was noted in any treatment group (100, 300 and 1000 mg/kg bw/day).
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).
A slightly increased salivation was noted in one male rat, no further signs of clinical toxicity were noted for the male and female rats of the high dose group (1000 mg/kg bw/day).
No test item related influence was noted for the male and female rats of all treatment groups (100, 300 and 1000 mg/kg bw/day) during the observational and functional (grip strength and spontaneous motility) neurological screenings.

BODY WEIGHT AND WEIGHT GAIN
A slight reduction in body weight was noted for the male and female rats of the high dose group (1000 mg/kg bw/day). For the male rats the reduction in body weight was noted from test day 8 (4.4%) until test day 36 (5.5%) and for the female rats from gestation day 0 (7.6%) until lactation day 4 (9.5%). The body weight at autopsy was reduced accordingly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
A slightly statistically significant (p ≤ 0.01) increase in relative food consumption by 10.3% was noted in the high dose males during the 2nd test week. This was caused by the reduced body weight of the rats of the high dose group.
No influence on food consumption was noted in any treatment group in the females.

HAEMATOLOGY
No test item-related influence was noted.

CLINICAL CHEMISTRY
The laboratory examinations revealed an increased ALAT activity for the male and female rats of the high dose group (1000 mg/kg bw/day) and a decrease in the albumin concentration for the male rats of the high dose group.

NEUROBEHAVIOUR
No test item-related influence was noted .

ORGAN WEIGHTS
No test item-related influence was noted .

GROSS PATHOLOGY
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 (1000 mg/kg bw/day) ), which were considered to be test item-related.
No test item related findings were noted in the female animals.

HISTOPATHOLOGY: NON-NEOPLASTIC (restricted to dose groups 1 and 4)
A statistically significant (p≤0.01) occurence of squamous cell hyperplasia in the non glandular mucosa of the forestomach was noted for the male and female rats (5 of 5 each) of the high dose group (1000 mg/kg bw/day). Occasionally the squamous cell hyperplasia with subsequent hyperkeratinization was associated with acute inflammation of the submucosa in the non-glandular stomach (for 2/5 males and 1/5 females).
A pulmonary congestion was found in 4 of 5 male animals, which was statistically significant (p≤0.05) in comparison to the control group (0/5).
No microscopic changes were noted for the reproductive organs of the male and female rats of the high dose group (1000 mg/kg bw/day).

Evaluation of reproduction parameters: see section 7.8.1 & 7.8.2

Dose descriptor:

NOAEL

Remarks:

Parental generation F0

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Systemic effects

Critical effects observed:

not specified

Table 1. Mean body weight males

Body Weight (g)
Sex: Male	Day(s) Relative to Start Date
	1	8v	15	22v	29v	36v
Group 1: control	293.02	334.59	350.28	386.70	410.83	440.86
Group 2: 100 mg/kg	293.46	328.96	347.75	380.93	405.63	436.65
Group 3: 300 mg/kg	294.16	336.32	353.72	392.53	416.39	446.90
Group 4: 1000 mg/kg	293.14	319.93*	338.13	367.75	389.07	416.42

Statistical Test Dunnett’s Test (Anova)

Group Factor Dunnett’s Test (Anova): v –Statistical Test: Analysis of Variance p<0.05

*-Statistical Test: Dunnett 2 Sided p<0.05

 

Table 2. Mean body weight females

Body Weight (g)
Sex: Female	Day(s) Relative to Start Date			Day(s) Relative to Mating (L)				Day(s) Relative to Littering (A)
	1	8	15	0	7v	14v	20	1vv	4v
Group 1: control	181.25	200.61	207.74	231.36	272.16	301.56	365.20	283.99	301.20
Group 2: 100 mg/kg	180.93	197.80	208.12	226.64	269.64	303.46	376.18	290.81	302.18
Group 3: 300 mg/kg	180.90	194.82	204.24	224.81	254.78	289.41	356.96	271.43	284.22
Group 4: 1000 mg/kg	181.33	193.29	200.78	213.67	245.74*	277.59*	341.34	258.20*	272.58*

Statistical Test Dunnett’s Test (Anova)

Group Factor Dunnett’s Test (Anova): v –Statistical Test: Analysis of Variance p<0.05

                                                                vv- Statistical Test: Analysis of Variance p<0.01

*-Statistical Test: Dunnett 2 Sided p<0.05

 

Table 3. Mean Biochemical Parameters Males and Females (Albumin and ALAT)

Biochemical Parameters
Sex: Male	Albumin (g/L)	ALAT (U/L)v	Sex: Female	Albumin (g/L)	ALAT (U/L)vv
Group 1: control	31.98	38.0	Group 1: control	33.14	38.4
Group 2: 100 mg/kg	31.64	38.6	Group 2: 100 mg/kg	32.80	36.4
Group 3: 300 mg/kg	31.18	43.4	Group 3: 300 mg/kg	33.74	34.6
Group 4: 1000 mg/kg	30.60**	63.2**	Group 4: 1000 mg/kg	32.18	56.2**

Statistical Test Dunnett’s Test (Anova)

Group Factor Dunnett’s Test (Anova): v –Statistical Test: Analysis of Variance p<0.05

                                                                vv- Statistical Test: Analysis of Variance p<0.01

* -Statistical Test: Dunnett 2 Sided p<0.05

** - Statistical Test: Dunnett 2 Sided p<0.01

  Table 4. Histopathology Males and Females

Sex	Male				Female
Group	Gr.1	Gr.2	Gr.3	Gr.4	Gr.1	Gr.2	Gr.3	Gr.4
Number of Animals	5			5	5			5
Number of Completed Animals	5			5	5			5
Lungs
congestion - slight - moderate - marked	0 0 0 0			4* 1 3 0	2 1 1 0			1 0 0 1
Stomach
No abnormalities detected	5			0	5			0
Non-glandular; submucosa; acute inflam-mation; focal -slight -moderate	0      0 0			2      1 1	0       0 0			1       0 1
Non-glandular; squamous cell hyperplasia -slight -moderate -marked	0   0 0 0			5**    1 3 1	0   0 0 0			5**    2 2 1
Non-glandular; keratopurulent debris	0			2	0			0

  Fisher’s Two-Tailed Exact Test Performed:

* = 5% Significance

** = 1% Significance

 

 

 

Conclusions:

NOAEL (no-observed-adverse-effect level) of the parental generation: 300 mg/kg bw/day, p.o.

Executive summary:

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 422. The test item was administered orally by gavage to rats at dose levels of 100, 300 and 1000 mg active ingredient/kg bw/day. The application started two weeks before mating on test day one and ended on the day or one day before sacrifice. Day of sacrifice was on test day 37 for the male rats and between lactation day 4 and 7 for the female rats.
No test item-related premature death was noted in any treatment group (100, 300 and 1000 mg/kg bw/day).

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). A slightly increased salivation was noted in one male rat, no further signs of clinical toxicity were noted for the male and female rats of the high dose group (1000 mg/kg bw/day). No test item related influence was noted for the male and female rats of all treatment groups (100, 300 and 1000 mg/kg bw/day) during the observational and functional (grip strength and spontaneous motility) neurological screenings. A slight reduction in body weight was noted for the male and female rats of the high dose group (1000 mg/kg bw/day). For the male rats the reduction in body weight was noted from test day 8 (4.4%) until test day 36 (5.5%) and for the female rats from gestation day 0 (7.6%) until lactation day 4 (9.5%). The body weight at autopsy was reduced accordingly.

The laboratory examinations revealed an increased ALAT activity for the male and female rats of the high dose group (1000 mg/kg bw/day) and a decrease in the 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 (1000 mg/kg bw/day). Microscopic examination revealed the occurrence of squamous cell hyperplasia in the non-glandular mucosa and acute inflamaation of the forestomach from the male and female rats of the high dose group (1000 mg test item/kg bw/day). Further microscopic findings occurred in form of pulmonary congestion in the male rats from the high dose group.
NOAEL (no-observed-adverse-effect level) for repeated dose toxicity: 300 mg/kg bw/day, p.o.

Effects on reproduction parameters and organs (see section 7.8.1).

Effects on the development of the F₁offsprings (pups) (see section 7.8.2).

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable quality (Klimisch2)

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

Only limied test data were available for current substance; most data were available from reac-across N2 and N3 subgroup members:

-'Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts'
-'Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium salts'.

Justification for read across within the category of N-containing sulphosuccinates (N2 and N3 subgroups) is documented in a separate document attached in Section 13.

Subacute toxicity
- A key study for repeated dose toxicity was therefore performed with ' 'Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18(evennumbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts' by means of an oral combined repeated dose and reproduction/development screening study according to OECD guideline 422 (Hansen, 2013a). 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.

- Supporting data were available for registered substance (test item containing 29.90% active ingredient) of a 14-day dose-range-finding in 5 male and 5 female rats treated once daily with 100, 300 or 1000 mg active ingredient/kg bw/day, by oral administration (Hansen, 2013a). None of the animals died prematurely or revealed any test item-related changes in behaviour, external appearance or faeces or changes in body weight, body weight gain, food and drinking water consumption as well as in relative and absolute organ weights. Macroscopic examination revealed no test item-related changes at any of the tested dose levels. NOAEL was 1000 mg/kg bw.
- Supporting data were also available for read across substance 'Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts' of a 14-day dose-range-finding study (Hansen, 2013c). 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. This substance was considered to be 'worst case' compared to the registered substance.

-In conclusion, NOAEL-levels of 300mg/kg bw was obtained in a combined repeated dose and reproductive/developmental screening study which was performed with read-across substance according to OECD guideline 422.

 

Subchronic toxicity
Data were available for read across substance 'Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium salts'.

- A supporting 14-day dose-range-finding was conducted with test item containing 34.4% active ingredient (Hansen, 2012d). Male and female rats were treated orally with 100, 300 or 1000 mg act. ing. /kg bw/day. No rats died prematurely nor revealed any test item-related changes in behaviour, external appearance or faeces. No changes in body weight and body weight gain, food and drinking water consumption or for relative and absolute organ weights were noted at any of the tested dose levels. Macroscopic examination revealed no test item-related changes at any of the tested dose levels. Maximum tolerated dose was 1000 mg/kg bw.
- In a key subchronic repeated dose toxicity study, 160 Sprague-Dawley albino rats, 80 of each sex, were fed a control diet, or 0.50, 2.00 or 8.00 g/kg /day of test item mixed in the diet (Tegeris and Underwood, 1976a). The liquid test item contained 35.8% active ingredient, which was taken into account for the dosages. The high dose was reduced to 4.00 g/kg/day mixed in the diet for weeks five through to termination. The study showed decreased body weight gain, feed consumption and food efficiency at the mid dose and high dose levels and increased SGOT and SGPT at the high dose. Further hematuria was seen in the mid and high dose rats, various organ weights were decreased (e.g. decrease in adrenal and gonadal weight in high dose groups; decrease in pituitary weight in females of high dose group) and lower urinary tract pathology was seen in 2 high dosed rats. It thus appeared that the NOEL in the rat was below 0.50 g/kg/day, however 0.5 g act.ingr./kg bw/day can be considered as NOAEL.

- In another subchronic repeated dose toxicity study, test item containing 35.8% active ingredient was given in the diet to purebred beagle dogs for ninety days (Tegeris and Underwood, 1976b). Thirty-two purebred beagle dogs, sixteen of each sex, with an average age of three to four months, were fed the control diet or 0.062, 0.250 or 1.000 g act.ingr. /kg bw/day thoroughly mixed in the diet. The dogs were carefully observed for the duration of the experiment and several hematological and biochemical parameters were conducted while the experiment was in progress. At the conclusion of the experiment all dogs were necropsied and all organs and tissues were examined histologically. These studies have shown that the test substance interfered with the average daily feed consumption of the mid-dose female and high dose test dogs and decreased the rate of bodyweight gain of the high dose test dogs when fed to them under the conditions of this experiment. Otherwise it was harmless up to 1 g/kg bw/day; a NOAEL of 0.250 g/kg bw/day can be considered.

 

General assessment and 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.
- Further information supporting the safety of the test substance is provided in the read across justification for the N2 and N3 subgroups, showing that all substances in these groups had similar NOAELs (justification with data matrix separately attached in Section 13).

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Key study

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; digestive: stomach

Justification for classification or non-classification

Based on these results and according to the EC Directive (No.93/21/EEC) and 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.